No title

Annual Review of Immunology Volume 5 1987

Before and After Elvin A. Kabat. Vol. 6: 1–25

Transgenic Mice and Oncogenesis Suzanne Cory and Jerry M. Adams. Vol. 6: 25–48

Kinin Formation: Mechanisms and Role in Inflammatory Disorders D Proud, and A P Kaplan. Vol. 6: 49–83

Immunobiology of CR2, the B Lymphocyte Receptor for Epstein-Barr Virus and the C3d Complement Fragment N R Cooper, M D Moore, and G R Nemerow. Vol. 6: 85–113

Veto Cells Pamela J. Fink, Richard P. Shimonkevitz, and Michael J. Bevan.Vol. 6: 115–137

Antigenic Variation in Lentiviral Diseases Janice E. Clements, Susan L. Gdovin, Ronald C. Montelaro, and Opendra Narayan.Vol. 6: 139–159

Structure, Organization, and Regulation of the Complement Genes R D Campbell, S K Alex Law, K B M Reid, and R B Sim. Vol. 6: 161–195

Normal, Autoimmune, and Malignant CD5+ B Cells: The LY-1 B Lineage? K Hayakawa, and R R Hardy. Vol. 6: 197–218

Opioid Peptides and Opioid Receptors in Cells of the Immune System N E S Sibinga, and A Goldstein. Vol. 6: 219–249

Structure and Function of Human and Murine Receptors for IgG J C Unkeless, E Scigliano, and V H Freedman. Vol. 6: 251–281

Melanoma Antigens: Immunological and Biological Characterization and Clinical Significance M Herlyn, and H Koprowski. Vol. 6: 283–308

The Developmental Biology of T Lymphocytes H V Boehmer. Vol. 6: 309–326

V Genes Encoding Autoantibodies: Molecular and Phenotypic Characteristics C A Bona. Vol. 6: 327–358

Role of the Major Histocompatibility Complex Class I Antigens in Tumor Growth and Metastasis K Tanaka, T Yoshioka, C Bieberich, and G Jay. Vol. 6: 359–380

The Immunoglobulin Superfamily—Domains for Cell Surface Recognition A F Williams, and A N Barclay. Vol. 6: 381–405

Lymphotoxin N L Paul, and N H Ruddle. Vol. 6: 407–438

Regulation of Cytokine Gene Expression T Taniguchi. Vol. 6: 439–464

Unique Tumor-Specific Antigens H Schreiber, P L Ward, D A Rowley, and H J Stauss. Vol. 6: 465–483

Molecular Regulation of B Lymphocyte Response T Kishimoto, and T Hirano. Vol. 6: 485–512

IgE-Binding Factors and Regulation of the IgE Antibody Response K Ishizaka. Vol. 6: 513–534

Nonprecipitating Asymmetric Antibodies R A Margni, and R A Binaghi. Vol. 6: 535–554

Three-Dimensional Structure of Antibodies P M Alzari, M B Lascombe, and R J Poljak. Vol. 6: 555–580

Prospects for Gene Therapy for Immunodeficiency Diseases P W Kantoff, S M Freeman, and W F Anderson. Vol. 6: 581–594

C1 Inhibitor and Hereditary Angioneurotic Edema A E Davis, III. Vol. 6: 595–628

The T Cell Receptor/CD3 Complex: A Dynamic Protein Ensemble H Clevers, B Alarcon, T Wileman, and C Terhorst. Vol. 6: 629–662

To the Malaria Circumsporozoite Protein: An Immunological Approach to Vaccine Development M F Good, J A Berzofsky, and L H Miller. Vol. 6: 663–688

Annu. Rev. Immunol. 1988.6:1-25. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Annual Reviews

Annual Reviews Ann.Rev. Immunol.1988.6: 1-24

IBEFORE AND AFTER

Annu. Rev. Immunol. 1988.6:1-25. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Elvin A. Kabat Departmentsof Microbiology, Genetics and Developmentand the Cancer Center/Institute for Cancer Research, ColumbiaUniversity College of Physicians and Surgeons, NewYork, NewYork 10032, and the National Institute of Allergyand InfectiousDiseases,NationalInstitutes of Health, Bethesda, Maryland20892 Introduction Theearlier portion of myautobiography(1) dealt with the period from the time I started to work with MichaelHeidelberger as a laboratory helpe~.on January 2, 1933 until mygrants from the USPublic Health Service weresummarilycancelled in 1952.This chapter continues mystory from that point. First though, I wouldlike to add a few notes about my origins andearliest days. Beginnings Mymother and father had married in 1913 and I was born on September 1, 1914, Both of myparents had cometo the United States as small children. Myfather, Harris Kabatchnick,wasborn in 1872and emigrated to the UnitedStates fromLithuaniaas a small boy. His first recollection after landing in the United States by boat from Hamburg was that the flags wereat half mastbecausePresidentGarfield hadjust died. His family settled on the lower East Side. Mymother, DoreenOtesky, cameto the UnitedStates from Kievin 1893at the age of seven. Myfather completed elementaryschool, went to work, and with his two brothers, Samueland Joseph, started manufacturing women’sdresses. In 1908 they changed their nameto Kabat and the firm wascalled KabatBros. I knowlittle of mymaternal grandfather. Hewaskilled in an accident before I wasborn. Mygrandmotherand the children lived with his brother Morris. It seemsthat the generationthat emigratesto a foreign country, andespeciallythe first generationbornin the UnitedStates, tends to ignore ~ The USGovernmenthas the right to retain a nonexclusive, royalty-free license in and to any copyright covering this paper.

Annual Reviews

Annu. Rev. Immunol. 1988.6:1-25. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

2 KABAT its history and background until a complete story becomes difficult or impossible to get. Mymother completed high school and played the piano very well. Her mother lived with us until her death, whenI was 11 or 12 years old. We had a five-room apartment on the fourth floor at 24 West 11 lth Street. Both my mother’s and father’s families were very close. Manylived in Harlem within four or five blocks of our home; others lived in the Bronx or in Brooklyn. Aunts, uncles, and their children visited one another frequently. Mycousin Saul Meylackson, a physician who had been a captain in the Medical Corps, US Army, during World War I, became my role model. Myinterest in medicine grew largely from him, and when he visited, as he and his wife Pearl did frequently, I constantly plied him with questions about his patients and about medical research. I used to visit his office frequently on Saturdays, especially during myhigh school and college years, and looked at the smears he made of urethral exudates to diagnose gonorrhea. Pearl’s brother was Dr. Murray Peshkin, a wellknownallergist at Mount Sinai hospital, whomI also questioned incessantly about medicine. One grewup in the 1910s and 1920s keenly aware of the role of infectious disease. I lost a brother whodied of pneumoniaat a few weeks of age in 1918; a cousin died of polio in the 1918 epidemic; myfather was very sick in the influenza pandemic of 1917; a friend in our apartment house died of diphtheria, and manyfamilies lost a child or youngrelative. Epidemics of whoopingcough, chicken pox, scarlet fever, measles, and diphtheria were frequent. Whenthe Schick test and immunization with diphtheria toxin-antitoxin were first introduced in NewYork City Schools in 1924, I was Schick negative, an early indication of mypotentiality as an antibody former. Myparents were very devoted to me and to mysister Harriet, born May 8, 1920. I had everything I wanted for the first 12 years of mylife. My mother tended to be somewhatover-protective. At the age of 10 or 11 I went to a school on 117th Street, and had to cross Lenoxand St. Nicholas Avenues on the way. She wanted to accompany me, but I absolutely refused. She then followed me at some discrete distance. WhenI turned around and saw her, I laid down in the middle of the road and motioned to her to go back before I would stand up. WhenI was seven or eight I went through a religious phase and asked mymother to get me a Hebrewteacher; I studied with him until I became Bar Mitzvah at 13. Unfortunately, I was taught to read and memorizebut never to knowthe structure of the language. This knowledgewould have been of great value in myextensive contacts with the WeizmannInstitute and Israel.

Annual Reviews

Annu. Rev. Immunol. 1988.6:1-25. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

BEFORE

AND AZI’ER

3

Myinterests in chemistry began early. Oneof myolder friends who lived on the samefloor wasgiven a chemistryset and let mehelp him. I soongot myownset and was alwaysexperimenting.Manyof the chemicals in the sets kids used in those days are nowhighly restricted even for laboratories. Myfather wouldstop off at Macy’son Saturday afternoon to buy mesomescience books.I wasgreatly influenced by Paul de Kruif’s MicrobeHunters. WhenI wasthirteen life becamevery difficult for us. Thefamily firm had prospered during the second and muchof the third decade of the century, manufacturingdresses selling for $24.95 to $89.95 wholesale. Thencheapdresses beganto flood the market, and the firm went bankrupt in 1927. They tried to get started again with moneyborrowed from relatives, but this enterprise also failed. From1929to 1933myfather did odd jobs, our incomedeclined continuously, and we were dispossessed from one apartment after another because we could not pay the rent. Whenwe movedinto a small apartment, wehad to put goods in storage and these were lost whenwe could not pay the monthlycharges. They containedmostof the papersof myearly life. Onelandlord turned off our electricity so we were in the dark. Wehad no food but a small piece of butter whichwekept from getting rancid by wrappingit and letting cold water run over it. Myfather and I went to court and the judge ordered the electricity turned on. Oneapartmenthotel held on to our furniture, so wethen had to find a furnished apartment. Twocousins whowere dress manufacturers,Arthurand Dick Shill, gave myfather a small job, helping to wait on customersin their showroom. Hestarted in 1932at five dollars per week, but as things improvedduring the NewDeal he continued to work and ended up with a more respectable salary--perhaps $40 to $50 per week. Mysurvival and ability to continue myeducation were largely consequencesof the educational concepts of the early 1920s. LewisM. Terman at Stanford during WorldWarI had emphasizedthe use of intelligence testing to select gifted children for special attention. TheNewYorkPublic SchoolSystemwasallowing bright children to skip grades (half years) they felt that they could do the work. WhenI entered elementaryschool in September1920, I wasalready able to read and to do arithmetic. This was in part becausethe kids in our apartmenthouse played school, with someof the older girls as teachers. Byskipping four times in elementary school I saved two years. WhenI entered DeWittClinton HighSchool at the age of 12, Terman’sextensive study of 1000gifted children whowere to be followedfor a goodportion of their lives waswell underway (2). The Termanwavehad reached high school. Bygetting goodgrades and taking somesummerclasses, I completedhigh school in three years instead of

Annual Reviews

Annu. Rev. Immunol. 1988.6:1-25. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

4

KABAT

four. Thus, in the fall of 1929, at the age of 15 I entered City College. I had applied for a Pulitzer Scholarship to Columbiabut was not selected. At City College I also did well. Since I received extra credits for A and B grades, applied toward the 128 required for the bachelors degree, and since I took one or two summercourses, I was again able to complete the four year course in three. By June of 1932, I was graduated from City College with a B.S. degree. I applied to two medical schools, Cornell and Columbia, whose catalogs said that they had scholarships. Indeed, Columbia had a scholarship specifically for a City College graduate, but it was not being awarded in those days. (It is of interest that I was subsequently on the medical faculty of both these institutions.) Regardless of the extensive subsequent changes in psychologists’ ideas about the limitations and validity of IQ tests, the Termanideas had a very crucial influence on what happened to me. From 1931 to the time I was graduated, the relatives who were helping to support us applied substantial pressure on meto quit college and get a job. I was picking up small amounts of moneyby working during college registration, and I worked as an usher in the old Loew’s NewYork movie theatre during one summer. However, the pressure kept growing for me to quit college. I finally told one of mymother’s brothers to "get the hell out and don’t come back." It was many years before we made up. Twogood friends, Joseph Silagy and Joel Hartley, graduated in 1931 and went to NewYork University Medical School. I used to visit them and sit in on their classes on Saturday mornings. I heard lectures by Homer Smith and R. Keith Carman. I later became very friendly with Cannan while he was at NewYork University as chairman of the Biochemistry Department and at the National Research Council. Wealso met during summers at Woods Hole. While at City College, I had the opportunity to work with Professor Leo Lehrmanin analytical chemistry. Later I started working with Michael Heidelberger, but I continued at City College on Tuesday and Thursday evenings, when Leo taught evening classes. Myearliest papers were published with him. I also becamevery friendly with the professor of organic chemistry, W. L. Prager, and with a biology instructor, Alexander S. Chaikelis. I took his very popular course in physiology during one summer. As a way of trying to help me financially, we constructed a large flow chart of synthetic reactions of aromatic compoundsand tried, unsuccessfully, to interest publishers in it. I did showit to MichaelHeidelberger while I was looking for a job; he liked it and pointed out sections that represented his earlier work. It probably influenced his decision to take me. Charts of this type later becamepopular teaching tools. I was a good Germanstudent and became very friendly with my German teacher, Mark Waldman. Leo

Annual Reviews BEFORE

AND AFTER

5

Annu. Rev. Immunol. 1988.6:1-25. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Lehrman tried, also unsuccessfully, for us to get the rights to translate Fritz Feigl’s bookon spot tests into English. I visited the Feigls many yearslater in Brazil. As a freshmanat City College, I brashly decided to volunteer for the curriculum committee, a student group that madesuggestions to the faculty. Theywere generally juniors and seniors; they had never had a freshmanon the committee,but they took me. I enjoyedtheir meetingsand did makesomesuggestions. DavidRittenberg was also on the committee duringmyfirst year. The Story Continues I will nowtake up mystory wherethe first chapter concluded. Onlyin 1981did I learn, underthe Freedomof InformationAct (Figures 1, 2), that the CriminalInvestigationDivisionof the Department of Justice had placed meon a "list for the apprehensionand detention of prominent individuals considered dangerousto the security of the UnitedStates." The FBI documentimplies that by 1954they were at least looking for information that might lead to myremovalfrom this Security Index. I include this since most Americansare probablyunawareof the existence of such a list. Considering the admittedinjustice of the forcedresettlement of Japanese Americansduring WorldWarII, one wonders howuseful sucha list actually is. Needlessto say, I wasneverapprehended or detained. The last monthsof 1953 and 1954were spent trying to continue work without support from the Public Health Service. Thestudies on cerebrospinal fluid gammaglobulin had proven very useful as an aid in the diagnosis of multiple sclerosis, and HoustonMerritt arranged for the Presbyterian Hospital to makeit a routine clinical laboratory deterruination. Theyprovidedmewith funds for a technician and someparttime help in washingglassware, drawnfromthe fees chargedfor the tests. My laboratory continued to do this until the late 1970s whenautomated methodsweresubstituted. I received a small grant from United Cerebral Palsy, whichhelped. I activated myONRcontract, whichallowed us to continue work on the blood group and dextran problems. The NSFgrant in 1954madeit possible to keepmytechnicians, graduateand postdoctoral students. The University took responsibility for mysalary (1). It was impossible, however,to support the monkeycolony; the allergic encephalomyelitis work was discontinued, just whenwe were planning to isolate the encephalitogenicantigen. Fortunately, this problemwastaken up by manyother workers. Blood Group Substances Weresumedworkon the structure of the BloodGroupA, B, H, Le", b, Le I and i glycoproteins. Mybook Blood GroupSubstances(3) was written

Annual Reviews KABAT

Annu. Rev. Immunol. 1988.6:1-25. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

6

Figure 1

during several summers at WoodsHole and was published in 1956. Oligosaccharides were isolated, after mild acid hydrolysis, and later by alkaline borohydride degradation. The use of periodate oxidation followed by Smith degradation made it possible to propose (with Kenneth O. Lloyd)

Annual Reviews

AND AFTER

7

Annu. Rev. Immunol. 1988.6:1-25. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

BEFORE

Figure 2

a composite structure for the carbohydrate moiety of the blood group substances. Later, DonCarlson developed improvedconditions that prevented peeling after alkaline elimination and madepossible isolation of oligosaccharides with reducedN-acetylgalactosaminitol, so that we could

Annual Reviews

Annu. Rev. Immunol. 1988.6:1-25. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

8

KABAT

isolate intact reduced oligosaccharide chains. Walter Morganand Winifred Watkins were isolating oligosaccharides and determining blood group structures independently during the same period. Our results were in excellent accord, and we becamevery close friends. Our oligosaccharides isolated by 3H-alkaline borotritiide differed extensively; manyside chains resulted from incomplete biosynthesis, and this revealed extensive heterogeneity. The findings up to 1982 have been extensively described in a more personalized account (4). Later studies with Albert M. Wu, David Zopf, and Bo Nilsson, Flavio Gruezo & Jerry Liao on six grams of a humanovarian cyst blood group A substance subjected to Smith & Carlson degradations yielded many more oligosaccharide structures from the interior of the blood group substances and made possible extension of the composite structure. This also permitted placing on the compositestructure the oligosaccharides isolated earlier from various intact blood group substances. These were done by Kenneth Lloyd, Luciana Rovis, Byron Anderson, Marilynn Etzler, and by Sherman Beychok,who also studied their optical rotatory dispersion and circular dichroism. The development of the hybridoma technique by Kohler & Milstein led many groups to produce monoclonal anti-A, anti-B, and other blood group reagents. Dr. H. T. Chen and I mappedthe fine structures of the combining sites of monoclonal anti-A and anti-B produced commercially in Canada, England, and Sweden. All were equivalent as blood grouping reagents by the usual hemagglutination tests, but their fine structures differed substantially. We have made cDNAsand have cloned and sequenced them to correlate sequence differences with variation in site structure. Interest in the blood group Ii system began with the studies of Donald Marcus and Richard Rosenfield in the early 1960s. These showed that enzymesof Clostridium tertium destroyed I activity of red ceils, liberating galactose and N-acetyl-glucosamine. Subsequent studies, with Ten Feizi, classified I and i antigens into groups. Anti-IMa(group 1) specificity was shown to involve DGalfl(l ~ 4)DGlcNAcfl(1 ~ OCH2-. With Ray Lemieux’s laboratory we have mapped the fine structure of monoclonal anti-IMa sites more extensively. Weused a substantial number of synthetic oligosaccharides, each modifying a distinct portion of the antigenic determinant, to evaluate its contribution to binding. Mostingenious was Lemieux’s replacement of one of the two H in the -OCH2moiety by deuterium to give two optical isomers, one of which was highly active and the other inactive. After leaving our laboratory, Dr. Feizi continued studies on other I and i determinants, and Dr. Marcusclarified the nature of the determinants of the blood group P system.

Annual Reviews

Annu. Rev. Immunol. 1988.6:1-25. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

BEFORE AND AFTER

9

Interlude The Josiah MacyFoundation ran a series of conferences on Polysaccharides in Biology during 1955-1959.Weall exchangedviews, using the Frank Fremont-Smithprinciple, "Don’t speak while I am interrupting." Oneamusingincident in 1955comesto mind. K. G. Stern and I differed on the question of whetherthe microheterogeneity of proteins wasascribable to mistakesin synthesis. I took the position that this could not be unequivocallyestablished since small amountsof impurities mightgive the appearanceof microheterogeneity. Stemreplied, "I cannothelp but feel that nature cannotworkso precisely that it can reproducea moleculeof a million or twentymillion molecularweightexactly in its own imageas, for example,the imprint of a gramophone record. Bywayof example,if two typewriters of the samemakeand the sametype are used, and the sameletters are writtenwiththem,at first glance,it will be said that the imprintis identical, but it is a well-knownfact that criminological investigation can distinguish betweendifferent individual typewriters by microscopicinvestigation, becausethere exist alwaysminute irregularities; so I wouldsay this is a microheterogeneity,somethingwhichat first approximationlooks homogeneous but cannotstand up to the mostsearching criteria." Boyd:"Youmeanthat nature does not always produce homogeneity,or can never produce homogeneity?" Stem:"I wouldsay that at the level of the maeromolecule, it is too muchto expect of nature that such structures shouldbe absolutelyidentical. This is a carry-overfrom the experiencewith small molecules." Kabat:"I think I wasvery careful to say that the evidencedid not permitus to infer whether or not nature produced a homogeneousmacromolceule.However,for the record I amperfectly willing to state that Dr. Stemhas completelyconvincedmeof the microheterogeneity of typewriters."

GeorgSpringerwhowasediting the conference,indexedthis under: "Typewriters, microheterogeneity of, p 163." Lectins I had beeninterested in lectins since the studies on ricin duringWorldWar II carried out with MichaelHeidelberger and AdaBezer. WhileI was in Swedenin 1967, Sten Hammarstrrm,from Peter Perlmann’s laboratory, proposed to becomea postdoctoral student. He mentionedthat he had isolated a lectin fromHelix pomatiawhichagglutinated A red cells, so I told himto bring somealong. Westudied its combiningsite and determined its association constant by equilibriumdialysis. MarilynnEtzler studied the site specificity of Dolichusbiflorus. ArneLundbladcamefromUpsala, bringing someof his blood group oligosaccharides fromurine whichwere of great help in mappingA, B, Hspecific combiningsites of lectins and antibodies.

Annual Reviews

Annu. Rev. Immunol. 1988.6:1-25. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

10

KABAT

WhenMiercio Pereira, with an M.D. degree, came to the laboratory from Brazil, he expandedthe lectin studies to purify and characterize the combiningsites of blood group specific lectins from Lotus, Ulex, soybean, and peanut. Hagen Bretting studied sponge lectins from Axinella; Jerzy Petryniak and Pereira studied Evonymuseuropeus; Shunji Sugii did l, Vistariafloribunda; and Paul Kaladas studied l~icia villosa with Kimuraand Erson from Hans Wigzell’s laboratory in Sweden. Hagen Bretting and Stephanie Phillips studied the mutageniceffects of,4xinella lectin. Charles Woodwith S. Ebisu, L. A. Murphy,and Irwin Goldstein investigated the lectins of Bandeireae simplicifolia A4, B4, and BSlI. Santosh Sikder with C. J. Steer and Gil Ashwellstudied the chicken hepatic lectin, and he did lima bean lectin with Goldstein and Dave Roberts. Wewere very fortunate to have collaborations with Nathan Sharon of the WeizmannInstitute and Irwin Goldstein at Ann Arbor and their colleagues on many of these studies. Manysimilarities exist between the fine structures of antibody combining sites to A, B, H glycoproteins and those of the blood group specific lectins as determined by immunochemicalmapping. Of special interest is the finding that lima bean lectin (5) and a hybridoma antiblood group (6) are very similar in reactivity with a monofucosylhexasaccharide from humanurine but behave entirely differently whena second fucose is substituted on the third sugar from the nonreducing end. The comparative mapping of the fine structures of lectin and antibody sites of similar specificities and correlating these differences with amino acid sequences and X-ray crystallographic structures should throw much light on the evolutionary emergence and functions of these two very important and diverse groups of molecules. Studies

on Antipolysaccharides

The immunochemical mapping of the combining sites and later recombinant DNAstudies on antibodies to ~(1 ~ 6)dextran became the predominant problem of the laboratory, with ancillary studies on anti-~(1 3)~(1 -~ 6)dextran (B1355S), antilevan, and anti-SIII. Rose Magestudied rabbit anti-or(1 ~ 6)dextran and could also showthat the rabbit anti-SIII site was complementaryto a hexasaccharide. Gerald Edelman studied the various antibodies I produced in myself--antidextran, antilevan, anti-A-as well as other humanantidextrans, after reduction and alkylation, using starch gel electrophoresis. These were studied in acrylamide, by Bill Yount and Marianne Dorner, and their subgroup compositions and genetic factors were determined by James Allen, Bill Yount, Marianne Dorner, and Henry Kunkel. Myantilevan turned out to be a monoclonal IgG; the others were pauciclonal. Aminoacid sequencing of antibodies was just

Annual Reviews

Annu. Rev. Immunol. 1988.6:1-25. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

BEFORE

AND AFTER 1 1

beginning. Hopingto be able to get enough of myantibodies to sequence, Dick Rosenfield kindly took 15 one-liter plasmaphereses from me, each a week apart, so that eventually I had about 7.5 liters of my serum to fractionate. Stuart Schlossman, Justus Gelzer, and Marianne Dorner tried to fractionate my antidextran to obtain monoclonal populations. While some fractionation into populations with smaller and larger antidextran sites was achieved, monoclonal populations were not obtained, and the amounts produced by fractionation were too small for the sequencing methods of the 1960s. Michael Potter at the National Institutes of Health and Melvin Cohn and Martin Weigert at the Salk Institute had isolated mouse monoclonal anti-~(1 -~ 6)dextrans. The fine structures of the combiningsites of three IgAs were studied with John Cisar, Jerry Liao, Marianne Dorner, and Michael Potter. In 1970 John Cisar had taken a summercourse that I gave at OregonState University at Corvallis, and he had cometo the lab after completing his PhDdegree. Weshowed that the combining sites of W3129 and QUPC52differed. W3129 and W3434were complementary to five ~(1 -~ 6) linked glucoses, whereas QUPC52was complementary to six. Despite this, the binding constant of QUPC52was only 1/30 that of W3129.By competition assays by equilibrium dialysis, methyl ~-D-glucoside and isomaltose contributed 60%of the binding energy of the pentasaccharide with W3129,but only 5%of the binding energy of the hexasaccharide with QUPC52.This indicated that the specificity of W3129 was directed toward the terminal nonreducing end of the ~(1 -* 6) linked oligosaccharide, whereas QUPC52 was specific for the internal chain of ¯ (1 -~ 6) linked glucoses, loosely termed cavity-type but also called end binders and groove-type sites, respectively. This distinction could be readily demonstrated with a synthetic linear dextran synthesized by Ruckel & Schuerch. Since the linear dextran had but one nonreducing end, it inhibited precipitation of W3129by dextran as well as the pentasaccharide; toward QUPC52,however, it was multivalent and so formed a precipitate with hybridomaascitic fluids. This provides a rapid screening methodfor classifying the two kinds of monoclonalanti-~(1 -~ 6)dextrans. Human Monoclonal

Antibodies

The demonstration by Waldenstr6m that macroglobulins and gamma globulins could occur as monoclonal homogeneousproteins in the serum of individuals with various blood dyscrasias, and by Edelmanand Gally that Bence Jones proteins were the light chains of immunoglobulins, led manyinvestigators to search for antibody activities..A numberwere soon reported. Several laboratories acquired large collections of humanmonoclonal proteins, but unlike the mousegammopathies, relatively few of

Annual Reviews

Annu. Rev. Immunol. 1988.6:1-25. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

12 KABAT the humanmonoclonalshad identifiable antibody activities. Myinterest in screening such humanmonoclonalsarose from two directions. Elliott Ossermanhad a large collection of sera from individuals with multiple myelomaand Waldenstrrm macroglobulinemia. Wedecided to screen these serums with mycollection of polysaccharides, blood group substances, etc. EdwardFranklin at NewYorkUniversity also had a large collection of humansera that had been screened for various antibody activities, but without success. MarianKoshlandhad asked him for a humanIgM;in an attempt to purify it over a Sepharosecolumn,it did not comeoff. It seemedto her that somemight be eluted with a number of sugars, so she thoughtI wouldbe interested. In screeningby gel diffusion we found that the IgMreacted strongly with a water extract of agar preparedat 20°C.Since agar contained4,6-pyruvylatedgalactose, I asked MichaelHeidelberger, whowas studying rabbit antisera to Klebsiella polysaccharides, for a numberof samples. The purified Franklin monowEAreacted muchmore strongly with Klebsiella K21than it clonal IgM did with agar in quantitative precipitin assays; only about one twentieth as muchof these Klebsiella K21polysaccharideswasneeded. At the same time we had found another humanmonoclonalIgM~AYin the Osserman collection that also reacted with agar and with the sameKlebsiella polysaccharide. Unlike IgMwEAMAy which was studied as purified IgM, IgM was studied using whole serum. Wetested other Klebsiella K polysaccharides, and K30and K33reacted in both sera in a wayidentical to K21.Onthe basis of these studies, whichhadbeen acceptedby the Journal of Experimental Medicine (7), the AmericanCancer Society gave me research development grant for a postdoctoral fellow, Dr. Arapalli S. Rao, whocamefromCalcutta. Since he wasto learn the quantitative precipitin methodto continue the work, I thought it wouldbe logical for him to MAy.Wehad run out of the first repeat the studies on IgMwEAand IgM sample of K21and madeup a solution from another sample. He learned the techniquerapidly and wasable to confirmall of our data except that K21gave no precipitate with IgMWEA; he becameconvincedthat our curve with K21waswrong.By this time the proof of the JEMpaper was due to arrive, and I had to decide whether to take out the curve of K21with wEA.In worrying about this I remembered IgM that MichaelHeidelberger had first sent mesix samples, of whichonly K21,whichhad the same4,6pyruvylatedgalactoseas agar, reacted, thus giving us our first clue as to wEA.Onlylater did we get K30and K33whichhad the specificity of IgM 3,4-pyruvylatedgalactose and precipitated equally well per unit weight. I told Raothat there must be someother explanation for the difference betweenthe two K21samples, since if the reaction with 1(21 had not providedthe initial clue to the specificities of the twoIgM,I wouldnever

Annual Reviews

Annu. Rev. Immunol. 1988.6:1-25. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

BEFORE AND AFTER

13

have been able to apply for the grant and he would not have come to the laboratory. The explanation finally emerged. It turned out that the first K21 was the neutral sodium salt, but the second K21 preparation had been isolated as the free acid and was progressively lowering the pHof the unbuffered purified IgMwEAbut was not altering the pH of the serum of IgMs~gv. Rao carried out a study of the effect of pH on IgMTM and IgMMAyand found that IgMwEggave identical precipitin curves with K21, sfgv K30, and K33 at pH 4, but K21 did not react at higher pH. With IgM all three polysaccharides reacted identically from pH7.0 to 4.0, indicating differences in the structures of the two antibody combiningsites. Subsequent screening of the Ossermancollection revealed a number of other humanIgM monoclonals with various anticarbohydrate specificities including antibodies specific for the interior of the water soluble blood group A and B substances and for chondroitin sulfates. Most exciting was the discovery of IgMN°v (8), an antibody specific for poly-~(2 ~ 8)Nacetylneuraminic acid, the specific capsular polysaccharide of the group B meningococcusand of E. coli K1, against which vaccination has not been successful in humans. This antibody is as protective per #g antibody in rats infected with E. coli as is a standard horse antigroup B meningococcal serum. IgM~°v also reacts with poly A, poly I, and with denatured DNA probably ascribable to similar oligomeric patterns of the carboxyls in the group B polysaccharide and the phosphates in the polynucleotides and in DNA,permitting reactivity in the IgMN°v antibody combining site. Michael Heidelberger in 1983 put forward a similar explanation of the cross-reaction of type 8 and 19 pneumococcalpolysaccharides; he suggested it was due to the ability of negatively charged phosphoryl-fl-D-Nacetylmannosamineto enter the type 8 site specific for cellobiuronic acid with its negatively charged COO-and vice versa. IgMN°v has potential as adjunct serotherapy together with standard antibiotic therapy for group B meningococcalmeningitis, a possibility suggested by studies in the first three decades of this century of reductions in the death rate due to intrathecal horse antimeningococcal serum. GiampaoloMerlini, Steve Birken, and Marian Gawinowiczare determining the amino acid sequence of the Vn and VL regions of IgMr~°v so that the antibody can be synthesized by recombinant DNAtechniques. Stanley Hoffman and Gerald Edelman have shown that IgMN°v is antibody to N-CAM,and Karl Pfenninger has found it to show the histochemical distribution of N-CAM. The Variability

Plot

Careful amino acid composition analyses of antibodies of different specificities by Marian Koshland had revealed differences. In 1965 Hilschmann and Craig presented the first amino acid sequences of two humanBence

Annual Reviews

Annu. Rev. Immunol. 1988.6:1-25. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

14 KABAT Jones proteins and showedthat they differed extensively in the first 107 residues fromthe aminoterminusbut wereessentially identical throughout the C-terminal half of the molecule. Theseare nowtermed variable and constant regions. At an antibody workshop(9) at the Weizmann Institute in 1965Dreyer & Bennettsuggestedthat these two domainswere codedby separate genes; additional sequences were presented by Putnam, Milstein, Hood, Gray, Dreyer, and their colleagues. I becameinterested in these developments and in 1966-1967began a Sabbatical in Pierre Grabar’s laboratory at the Institut Pasteur, writing the first edition of "Structural Conceptsin Immunology and Immunochemistry."I thought it would be important to include the sequencedata. At that time two mousex and several human k BenceJones sequenceshad been published. Onaligning the data it was evident that in variable regions of humanand mousevery few aminoacid differencesdistinguish mousefromhuman,e.g. there werevery few speciesspecific residues (nowtermed"species-associatedresidues," as later suggested by Capra). The constant region, however,contained43 differences between humanand mouse; this was comparable to findings with hemoglobins, cytochromes,etc, from different species. A sequenceof a human 2 Bence Jones protein by Titani, Wikler, and Putnamappeared, which further reducedthe numberof species-associated residues and was cited in a "note added in proof." Francois Jacob and VernonIngram both thought the finding important, so I sent it to the Proceedingsof the NationalAcademy of Sciences. Shortly thereafter, in comparingthe V and C regions, I noted the occurrence of moreinvariant glycines in the Vregion, including those at residues 99 and 101 whichhavenowbeenfound in homologouspositions of V-regionsin essentially all immunoglobulin light and heavychains, in the a and fl chains of the T-cell receptor for antigen, andin T cell surface antigens T4andT8. I suggestedthat the role of these two variant glycines mightbe to provide flexibility and permit movementof the rest of the V-region to makemaximum contact with an antigenic determinant. Cesar Milstein, Niall & Edman, and the Hood group published sequencesof several V~chains whichindicated that there weresubgroups largely based on the first 23 aminoacids from the N-terminus. It then becameevident fromthe frequencyof identical repeats that this segment of the chain did not showthe variability neededfor it to be involved in generating antibody complementarityand diversity. This led to my suggesting that the first 23 aminoacids were involved only in threedimensionalfolding. Also, Milstein had noticed that beyondaminoacid 94 he could no longer define subgroupsand suggestedthat frequent crossing over mightbe occurring beyondresidue 94, a finding prophetic of V-

Annual Reviews

Annu. Rev. Immunol. 1988.6:1-25. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

BEFORE AND AFTER

15

J joining, which was largely ignored. The 1967 Cold Spring Harbor and Nobel Symposia provided forums for these exciting developments on aminoacid sequences of antibodies. In 1969 I received a letter from a Dr. Tai Te Wu, then at Cornell University Medical College and Sloan-Kettering in NewYork, asking if he could spend some time in the laboratory to learn quantitative immunochemistry. Whenhe visited the laboratory I asked about his background. He said he was a mathematical biophysicist and had some computer experience. I proposed that we collaborate on the sequence variability. I had previously been entering and aligning the sequence data by hand using pencil and paper. Webegan to meet one day a week to evaluate and discuss results. By this time more complete sequences were beginning to pour into the literature from the laboratories of Capra, Edelman, Hilschmann, Hood, Metzger, Potter, Milstein, and their coworkers. In June 1969 at a symposiumin Prague, Edelman, Franek, and I discussed positions containing more substitutions than were seen in the subgroups. Franek had tabulated dissimilarity in the subgroups, whereas Wu and I had searched for maximumvariability, and our data were in good agreement for three hypervariable regions. In June 1970 (10) we introduced an equation measuringvariability at each position in a set of VLsequences aligned for maximumhomology as follows: Numberof different aminoacids at a given position Variability = Frequencyof the most common aminoacid at that position" At this time some 77 complete and partial sequences of VLregions of humanV~ and V~ and of mouse V~ chains were available. This led to the recognition of three linear stretches of hypervariability, whichwe predicted would fold to form the antibody combiningsite. Whena sufficient number of VHsequences were examined, they too had three hypervariable regions (11). X-ray crystallographic studies on Fab fragments, BenceJones dimers, and more recently on the lysozyme-antilysozyme site by the Poljak group at the Institut Pasteur have all confirmed this prediction almost on a residue-by-residue basis. These hypervariable regions are nowtermed complementarity-determining regions (CDR). Wuand I proposed (10) in an insertional mechanismby which nucleotides coding for the CDRswould be recombined into the frameworkresidues of the various subgroups. The variability plot was based on the assumptionthat, since the antibody forming system was universal among vertebrates, combining data from different subgroups, species, etc, was justifiable, and the combiningsites would be in one place rather than in different portions of the variable regions. WhenMartin Weigert and Melvin Cohn with Cesari and Yon-

Annual Reviews

Annu. Rev. Immunol. 1988.6:1-25. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

16 KABAT kevich described the mouseV~chains, they found that most of their sequenceswere identical throughoutthe Vxregion. Theypostulated that these werethe productof a single germ-linegene. Thosechains that showed variation from the germ-line sequence were localized to the CDRsand could have been formedby point mutations. This providedthe first evidence for somatic mutation in immunoglobulinV~regions. The assumption is still valid for the very large numberof VLand VHchains now available. Variability plots havesince beenusedto define better allelisms in the MHC class I and II systemsand also to locate sites of variability in AIDS viruses. Variability plots for the T cell receptor require further analysis. Variability plots for other proteins, such as cytochromes,did not show hypervariable regions. A statistical examinationof each residue in the CDRsmadeit possible to designate certain positions as structural and others as probablyin contact with the antigenic determinant.

Compilationof Sequencesof ImmunoglobulinChains In 1973Harry Rose retired as chairmanof the MicrobiologyDepartment, and Harold S. Ginsburg whomI knew very well from WoodsHole was appointed.Since he wasjust getting started, I delayedmySabbatical one year so that I could participate in the teaching. I waschosenas a Fogarty Scholar and spent 1974-1975 at the NationalInstitutes of Health, writing the second edition of Structural Concepts in Immunologyand Immunochemistry. As it is customaryfor Fogarty Scholars to meet with the director of NIH,I was given an appointmentwith Dr. Robert S. Stone, whoinquired as to myresearch interests. WhenI replied that I was interested in the structure of antibody combiningsites and had been tabulating variable region sequences, he suggestedthat NIHhad the PROPr~Tcomputersystem and that this might help me. DeWittStettin, Jr., then DeputyDirector for Science at NIH, introduced me to WilliamF. Raub, then with the Division of Research Resources. NowDeputyDirector of NIH,he took a great interest in myactivity. To evaluate PROPrmT, HowardBilofsky came downfrom Bolt Beranek and Newman to help me in preparingthe tables of sequencesto be used in Structural Concepts.It soonbecameclear that PROPI-IEX had greatly superior potentialities for tabulating and keepingtrack of variable region sequences. Whenmyterm as a Fogarty Scholar was up, I wasasked to serve as an Expert, first to the NationalCancerInstitute andlater to the NationalInstitute of Allergy and Infectious Disease, spendingtwo days a weekat NIHto organize and maintain a data bank of variable region sequences.I rented an apartment in Building 20 at NIH. T. T. Wucame from Northwestern and Howard Bilofsky from Cambridgeto spend two days a monthkeeping track of and

Annual Reviews

Annu. Rev. Immunol. 1988.6:1-25. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

BEFORE AND AFTER

17

checkingthe data that I collectedfromthe literature. Thefirst edition (12) appeared in 1976 and contained only aminoacid sequences of variable regions; the secondedition (13) wasexpandedto include constant regions; the third (14) also containednucleotide sequencesand derived aminoacid sequences and included other membersof the immunoglobulinsuperfamily. Thefourth edition (15) appearedin April 1987, included T cell receptors, and had grownto 804 pages; Wuand I, along with Harold M. Perry and KayGottesman,are nowworkingon a fifth edition. Keeping track of exponentiallyincreasingdata is extraordinarilydifficult. Thecompendiumhas been distributed free to workers in the field and has been consideredvery useful. WhileI amvery happythat so manycolleagues find it valuable, I would not have undertaken it had I not felt it was essential to myresearch interests. In addition to providing an enormousbodyof data supporting our prediction (10, 11) that the CDRs in the light and heavychains would fold up to form the antibody combiningsite, wewere able to demonstrate independentassortment of framework(FR) aminoacids (16). This led to formulate the minigenehypothesis, in whichnucleotides coding for the three CDRswere assorted by recombination or insertion into the frameworknucleotides. This hypothesis was put forwardjust as Tonegawa et al. (17) had shownthat, in 12-day-old mouseembryoDNA,the region nucleotides only codedthrough aminoacid 95. Since adult myeloma coded through amino acid 107, we postulated a mechanismof somatic joining of a minigenethat codedfor the remainingaminoacid to give the intact V gene. The J minigene coding for amino acids 96 to 107 was found shortly thereafter by Tonegawa in 12-day-old embryoDNA.In the expressed assembledgene from adult myelomaDNA,it had been joined to the V-region(18). D and J minigenescoding for segmentsof the genewerefoundshortly thereafter (19). In the T-cell receptor for antigen, J minigenesare also present in e, fl, and ~ chains and Dminigenesin the fl chains. Thesefindingshold for all species examined. Since genescodingfor the rest of the VLand VHoccurredin the germline as single stretches of nucleotides, considerablecontroversyexisted as to whetherthe minigenehypothesisapplied to the V-region. DavidBaltimore and RichardEgel proposedthat geneconversionscould explain our assortmentdata. The Rajewskylaboratory (20) described a double recombinant in which CDR1of one germline gene was recombined with CDR2and 3 from another. H. G. Zachau’s laboratory (21) showedby nucleotide sequencingand comparisonsof V-regionsthat our assortment data could be accounted for by gene conversions in the DNAcoding for the FR segments.Mostrelevant are the studies of Reynaudet al (22) in J. Weill’s laboratoryat the Institut Pasteuron chickenVachains. Thechicken

Annual Reviews

18 KABAT

Annu. Rev. Immunol. 1988.6:1-25. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

has only a single functional germline V~gene and a large numberof pseudogenes;thus diversity andthe antibody repertoire must be generated by rescue of functional CDRsof pseudogenes by gene conversions-essentially our original minigenehypothesis(16). Work with WHO In 1963, the WorldHealth Organizationbecameinterested in research and training in immunology. Theyorganizedfive meetingsof scientific groups. I wasinvited to participate at one of these at Ibadanin December 1964. Niels Jerne was one of the prime movers.I met Professor Ian Mclntyre, then Deanof the Faculty of Veterinary Science at University College, Nairobi, and Professor and later Dean in the Veterinary School at Glasgow.Webecomeclose friends. Ahigh priority wasassigned to setting up research and training centers in immunology in developingcountries in various parts of the world. Thesewereto be coordinatedwith centers in Geneva and elsewhere. HowardGoodman,Zdenek Trnka, and I went to Nigeria, Uganda,and Gambia.Weselected Ibadanas the first center. Ada E. Bezer, whohad been a technician with me for manyyears, and Dr. Ivan Riha fromPraguestaffed the center, whichwasin the Department of ChemicalPathology.A course in Immunology wasgiven for six to eight monthsfollowedby opportunities for students to do research. Thecenters initially took students fromall of WestAfrica and a numberof students completed PhDs. In 1966 HowardGoodmanand I went around the world and selected Singaporeas another center. Centerswereultimately selected in Sao Paolo, MexicoCity, Nairobi, Beirut, Teheran,and NewDelhi, with support centers in Lausanneand Geneva.From1968to 1971, I spent two weekseachyear (the first three years) with Dr. Otto G. Bier, andthe last year with lvam Mota, directors at the Sao Paolo center located at the Escola Paolista and later movedto the Institute Butantan. I conducteda reviewof the coursework, gavethe final exam,and tried to get a regular research seminarstarted. It wasvery difficult to maintaina critical mass and sustained enthusiasm for continuing the seminars whenI was not there. Dr. VasekHoubaand AdaBezer staffed the Nairobi center for several years. Later, regional centers tended to drawstudents moreexclusively from the country in whichthey were located, and Thailandset up its own immunological center. Dr. GeorgioTorrigiani is currently in chargeof the program,but WHO has difficulties supportingthe centers (23). I continue as a memberof the WHO Expert Advisory Panel on Immunologyand have recently been involved with work on immunizationwith bacterial polysaeeharides. A further developmentwas the setting up of ILRAD (International Laboratoryfor Researchin AnimalDiseases) in Nairobi.

Annual Reviews BEFORE

AND AFTER

19

This began work in 1978 under the auspices of the WorldBank, the Rockefeller Foundation, and the Agencyfor International Development, to develop vaccines against east coast fever and trypanosomiasis. I was involvedin the early planning.

Annu. Rev. Immunol. 1988.6:1-25. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Interlude OnDecember26, 1970, the front page of the NewYork Times contained an appealfrom 14 Soviet scientists to President Nixonto see that Angela Davisreceiveda fair trial. I thought:"Wehavea free judicial system,the trial will be open to the public; President Nixonoughtto invite themto comeand see for themselves."I reflected on howone mightget himto do this. It became clear that onecould not write to the Presidentdirectly; he wouldnever see the letter. I decidedthat one hadto addressthe suggestion to someone on his staff, whoat least could bring it to his attention if he thoughtit a goodidea. I sent the followingtelegram: Dec. 26, 1970 John Ehrlichman The White House Suggest President Nixon reply to fourteen Soviet scientists’ appeal about Angela Davis by inviting them or any Soviet lawyers they nominate to attend her trial. Stop. Also call attention to request of five NewYork City District Attorneys to attend trials of Jews accused by Soviet Union of plotting highjacking. Elvin A. Kabat Member, National Academyof Sciences

Theidea evidently was favorably received. Figure 3 is the reply from John Ehrlichman.On Sunday,January 3, 1971, the six o’clock evening newsannouncedthat President Nixonhad invited the 14 Soviet scientists to attend AngelaDavis’s trial. Figure 4 showsa portion of Monday’s New YorkTimesarticle andits editorial of January8. It still seemsremarkable that a suggestionfroma private individual could reach the highest levels and be acted uponso rapidly. Current Activities and Future Plans AlthoughI becameEmerituson December31, 1984, I have been permitted to keep mylaboratories and to maintain mygroup at about the samesize. I decideda few years backthat the laboratory had to go into cloning and sequencingif wewereever to be able to formulatea detailed conceptof the interactions in the CDRsof antibodycombiningsites to explain antibody diversity. AccordinglyI spent a short time in MarkDavis’ laboratory, then at NIH, learning to makeand done cDNA.This was followed by about 10 days at the Weizmann Institut learning nucleotide sequencingin DavidGivol’s laboratory. I workedclosely with the carbohydratechemists

Annual Reviews

20 KABAT

1970

Annu. Rev. Immunol. 1988.6:1-25. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Dear M3:. Kabob: Thanks very much for your telegram in which ~’ou put fo~h your thoughts Presidenttsreply to the appeal ~de So%e% se~tis~s ~ be~lf Of ~gela

of December Z7th regarding ~e by fourteen Da~s.

i am sendln~ your wire to Henry Kissingerfor his

Thanks again

for letting

me have your thoughts.

7{) i{ave~Avenue New York 10032

Fiyure 3

in the laboratory,SantoshSikderandPradipAkolkar,to establish the making,cloning, andsequencingof cDNAs on a routine basis. Theythen taughtthe otherpost-doctorals,graduatestudents,andtechnicians. Weset ourselvesthe goal of trying to probethe repertoireof antibody sites formedto a single antigenicdeterminant, the epitope of a(1 -, 6)linked glucoses fromdextranB512.Graduatestudents JacquelineSharon and BarbaraNewman had been engaged in mappingthe antibody combiningsites ofhybridoma antibodiespreparedby injecting ~(1 -~ 6)dextran

Annual Reviews

Annu. Rev. Immunol. 1988.6:1-25. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

BEFORE ANDAFTER 21

Fiyure4 into BALB/cand C57BL/6mice. Eric Lai had carried out similar studies of C57BL/6hybridom~is from mice immunizedwith stearylisomaltosyl oligosaccharides. H. T. Chenand D. Makovercharacterized hybridomas to the stearylisomaltosyl oligosaccharides in a nude mouseand in C58 mice. Thus, we had a sampling of hybridomas with groove-type sites complementaryto six and seven ~(1 ~ 6)-linked glucoses. These were about equally divided between IgM and IgA, but two IgG3 hybridomas

Annual Reviews

Annu. Rev. Immunol. 1988.6:1-25. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

22 r,~d~Ar were obtained. Both antigens used for immunizationyielded T-independent hybridomas. Flavio Gruezo prepared mRNA and Drs. Sikder, Akolkar, Bhattacharya, and Jerry Liao have been cloning and sequencing them. Sherie Morrisonis collaborating. She and her colleagues pioneered in preparing transfectomassubstituting C-regiongenes of other species onto mouseVngenes. WithAmeliaBlack, a graduate student, Sherie and I are trying to express the transfection products in E. coli and in mammaliancells. TsukasaMatsudais studyingthe repertoire by the use of the isomaltose oligosaccharides coupledto BSAor to KLHto obtain a set of hybridomasto T-dependentantigens with an ~(1 ~ 6) epitope sufficiently large to fill the antibodycombiningsite. Paula Bordenhas preparedantiidiotypic hybridomasand is studying the idiotypic determinants of the various anti-a(1 ~ 6)dextrans. Oneimportant finding is that the T-independenthybridomashave Via chains belonging to three major germline gene families of Brodeurand Riblet, J558, J606, and J36-60and that the light chains also belong to several germline gene families although their combiningsites are very similar. This wouldtend to indicate that the antibody-formingsystemis very well protected against germlinegeneloss. Since mostother epitopes prepared by coupling small moleculesto a protein create heterogeneous populationsof antibodies, the findingthat different germlinegenefamilies were used could not be interpreted unambiguously. Paula Bordenfound that IgG3antiidiotypes to an anti-a(1 ~ 6)dextran hybridomaprecipitated with antidextran. The reaction differs from the usual precipitin reactions in that it is highly dissociated, and its role in idiotype antiidiotype reactions requires further study. H.-T. Chenhas mappedthe combiningsites of various antiblood group A and B hybridomas and, with Arne Lundblad and R. M. Ratcliffe, has cloned and sequencedseveral. These blood group epitopes are more complicatedstructurally and should also provide an intimate picture of those antibody combiningsites. Wesorely need high resolution X-ray studies of one or more antibody combiningsites to carbohydrate epitopes to be able to correlate immunochemicalmappingdata with the interactions of the dextran or blood groupoligosaccharidesin their respective sites. Wealso intend to study anti-antiidiotypes in this systemin relation to the "internal image"antibodyto a carbohydrate.I intend to stay in the laboratoryfor as long as I feel I can do creative work. Mylife at Columbiahas alwaysbeen most satisfying. Overthe years I have been able to collaborate with manycolleagues at Columbiaand throughout the world. The University provided mewith the opportunity to do as I wishedscientifically and alwaysdefendedacademicfreedomin

Annual Reviews BEFORE AND AFTER

23

general and mine in particular. I am indebted to the Office of Naval Research for seventeen years of support; to the molecular biology section of the National Science Foundation for 36 years of continuous support; and since 1974 to the National Institutes of Health for providing the opportunity for me to pursue my interests there as well as at Columbia. I thank Mr. Darryl J. Guinyard for typing the manuscript.

Annu. Rev. Immunol. 1988.6:1-25. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Literature Cited I. Kabat, E. A. 1983. Getting started 50 years ago--Experiences,prospectives, andproblemsof the first 21 years. Ann. Rev. Immunol.l: 1-32 2. Boring, E. G. 1956. LewisMadisonTerman.FromBiographical Memoirsof the NationalAcademy of Sciences1959, Vol. 33: 414-61. NewYork: ColumbiaUniv. Press 3. Kabat, E. A. 1956. Blood GroupSubstances--Their Chemistry and Immunochemistry. NewYork: Academic 4. Kabat, E. A. 1982. Contributions of quantitative immunochemistry to knowledge of blood groupA, B, H, Le, I and i antigens. Am.J. Clin. Pathol.78: 28192 5. Sikder, S. K., Kabat,E. A., Roberts,D. D., Goldstein, I. J. 1986. Immunochemicalstudies on the combiningsite of the blood groupA-specific lima bean leetin. Carbohyd.Res. 151:247~i0 6. Chen,H.-T., Kabat, E. A. 1985. Immunochemical studies on blood groups. Thecombining site specificities of mouse monoclonalhybrldomaanti-A and antiB. J. Biol. Chem.260:13208-17 7. Kabat, E. A., Liao, J., Bretting, H., Franklin,E. C., Geltner, D., Frangione, B., Koshland,M.E., Shyong,J., Osserman, E. F. 1980. Humanmonoclonal maerogiobulins withspecificity for Klebsiella K polysaccharides that contain 3,4-pyruvylated-D-galactose and 4,6pyruvylated-D-galactose.J. Exp. Med. 152:979-95 8. Kabat, E. A., Niekerson, K. G., Liao, J., Grossbard, L., Osserman,E. F., Glickman,E., Chess,L., Robbins,J. B., Schneerson, R., Yang, Y. 1986. A humanmonoclonalmacroglobulin with specificity for ~z(1 ~ 8)-linked poly-Nacetyl neuraminic acid, the capsular polysaccharideof groupB meningococci and Escherichiacoli K1, whichcrossreacts with polynucleotides and with denatured DNA.J. Exp. Med.164: 64254 9. Porter, R. R. 1986. Antibodystructure

and the antibody workshop1958-1965. Symposiumon The Role and Significance of International Cooperationin the BiomedicalSciences,eds. G. Salvatore, H. K. Schachman,in Perspectives in Biology and Medicine29: Part 2, S161-S165 10. Wu,T. T., Kabat,E. A. 1970. Ananalysis of the sequences of the variable regions of Bence Jones proteins and myelomalight chains and their implications for antibody complementarity. J. Exp. Med. 132:211-50 11. Kabat, E. A., Wu,T. T. 1972. Attempts to locate complementarity-determining residuesin the variablepositionsof light and heavy chains. Ann. N.Y. Acad. Sci. 190:382-93 12. Kabat, E. A., Wu,T. T., Bilofsky, H. 1976. Variable regions of immunoglobulin chains. MedicalComputerSystems. Cambridge,MA:Bolt Beranek & Newman 13. Kabat, E. A., Wu,T. T.~ Bilofsky, H. 1979. Sequences of Immunoglobulin Chains. NIHPublication 80-2008. National Institutes of Health, Bethesda, Md. 14. Kabat,E. A., Wu,T. T., Bilofsky, H., Reid-Miller, M., Perry, H. 1983. Sequences of Proteins of Immunological Interest. NationalInstitutes of Health, Bethesda, Md. 15. Kabat, E. A., Wu,T. T., Reid-Miller, M., Perry, H. M., Gottesman, K. S. 1987. Sequencesof Proteins of Immunological Interest. U.S. Departmentof Health and HumanServices, Public Health Service, National Institutes of Health, No. 165-462,pp. 1-804. Washington, DC: USGPO 16. Kabat, E. A., Wu,T. T., Bilofsky, H. 1978. Variable region genes for the immunoglobulinframeworkare assembled from small segments of DNA--A hypothesis. Proc. Natl. Acad.Sci. USA 75:2429-33 17. Tonegawa,S., Maxam,A. M., Tizard, R., Bernard, O., Gilbert, W. 1978.

Annual Reviews

Annu. Rev. Immunol. 1988.6:1-25. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

24 roa~,a" Sequenceof a mousegerm-linegene for version. Proc.Natl. Acad.Sci. USA80: a variable region of an immunoglobulin 4997-5001 light chain. Proc. Natl. Acad.Sci. USA 21. Jaenichen, H.-R., Pech, M., Linden75:1485-89 maier, W., Wildgruber,N., Zachau,H. 18. Bernard, O., Hozumi,N., Tonegawa,S. G. 1984. CompositehumanVkgenes and 1978. Sequences of mouse immunoa modelof their evolution.NucleicAcids globulin light chain genes before and Res. 12:5249-63 after somatic changes. Cell 15: 1133- 22. Reynaud, C.-A., Anquez,V., Grimal, 40 H., Weill, J.-C. 1987. Ahyperconversion 19. Early, P., Huang, H., Davis, M., mechanism generates the chicken light chain preimmunerepertoire. Cell 48: Calame, K., Hood, L. 1980. An immunoglobulinheavychain variable region 379-88 geneis generatedfromthree segmentsof 23. Kabat,E. A. 1986.A tradition of interDNA:V~, D, and J~. Cell 19:981-92 national cooperation in immunology. 20. Krawinkel, U., Zoebelein, G., BrugProc. Int. Symp. "The Role and Si#gemann,M., Radbruch, A., Rajewsky, nifieance of lnternationalCooperation in K. 1983. Recombinationbetween antithe Biomedical Sciences,"" ed. G.Salvabody heavy-chain V and variable V tore, H. K. Schachman.Perspect. Biol. region genes: Evidencefor gene conMed. 29:S159-S160

Annu. Rev. Immunol. 1988.6:1-25. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Annu. Rev. Immunol. 1988.6:1-25. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Annual Reviews www.annualreviews.org/aronline Ann. Rev. Immunol. 1988. 6 : 25-48 Copyright © 1988 by Annual Reviews Inc. All rights reserved

Annu. Rev. Immunol. 1988.6:25-48. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

TRANSGENIC MICE AND ONCOGENESIS Suzanne Cory and Jerry M. Adams TheWalterand Eliza Hall Institute of MedicalResearch,Post Office Royal MelbourneHospital, Victoria, 3050, Australia INTRODUCTION Thecreation of transgenic mice carrying specific cancer-promotinggenes has openedan exciting newera in oncology.Thebiological effects of an individual oncogeneon diverse cell types can nowbe assessed directly within the living animal. Whiletransgenic animals bear the introduced oncogenein every tissue, expressionof that genemayeither be widespread or directed to a particular cell lineage, dependinguponthe regulatory sequenceschosen, Thetransgene shouldbehaveidentically in every animal of an establishedlineageand, indeed, perhapsin everycell of a giventype. Thus, a well-characterized transgenic line becomesa permanentresource. Perhapsthe most significant opportunity providedby these transgenic animalsis the possibility of exploring the pre-neoplastic state. Onecan attempt to assess whetheran oncogenehas perturbeddifferentiation within particular lineages. The perturbations mayhelp to delineate early maturation stages and to clarify howdifferentiation is controlled. Thus,new insights mayemergeregarding the normalbiological functions of protooncogenes. The rules for oncogenecooperativity can also be evaluated within diverse cell types. For example,onecan isolate the relevant cells from a pre-neoplastic animal bearing an oncogeneand attempt to transformthemfully in vitro with retroviruses carrying other oncogenes.Alternatively, the second oncogenecould be introduced simply by breeding mice of two independenttransgenic lines. Whilethe studyof transgeniconcogenes is still in its infancy, majornew insights have already been gained. This chapter briefly summarizeshow transgenic animalsare produced,and then considers our current state of 25 0732~582/88/04104)025502.00

Annual Reviews 26

CORY & ADAMS

knowledgeconcerningthe effects of the different classes of oncogenesin transgenic mice. PRODUCTION

OF TRANSGENIC

MICE

Annu. Rev. Immunol. 1988.6:25-48. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Gene Transfer to the Germline by Microinjection Introduction of newgenes into the mousegermlineis currently performed most efficiently by microinjection of DNA.Onlya brief outline of the technologyis presented here, since detailed reviews haveappearedelsewhere(1-5). Pronucleiof recently fertilized eggs are injected with a few hundredcopies of the cloned gene of interest, and the embryosare then transferred into the oviducts of pseudo-pregnantfemales and allowedto develop to term. In skilled hands, some25%of the mice that are born carry one or morecopies of the foreign gene. If integration occursprior to the first cell division,all cells in a transgenicpup,includingthe germcells, will carry the "transgene."In practice, a significant proportion(~ 30%) the transgenic pups are mosaic, indicating that integration often occurs subsequentto the first round of replication. Since the same degree of mosaicismis usually exhibited in somatic and germcells, lines reliably transmitting the transgene can usually be established even from mosaic foundersby breedingtheir transgenic offspring. The mechanismof integration is not known.Presumably on random breakage of a mousechromosome,the injected DNA is incorporated by the normalcellular repair enzymes(for a discussion, see 4). Typically, some1-50 copies of the injected gene are inserted in a tandem,head-totail array at a single chromosomalsite. On subsequent breeding, the transgenic array is inherited as a simple Mendeliantrait, usually in a remarkablystable manner.Morerarely, insertion occursat two positions, and these loci segregate amongthe progeny.Since the transgeneis inserted into only one homologueof the relevant chromosome, the founder mouse is heterozygousat that allele. Homozygous lines can be established by breedingonly if insertion has not interrupted a genenecessaryfor development(6). Expression of an inserted gene depends primarily on the regulatory regions that it bears. A majordeterminantis "strength" of the promoter/ enhancercombinationused, but the level and tissue distribution of expression mayalso be influencedby the site of chromosomal insertion and perhaps by unknownelements within the transgene, such as attachment sites for the nuclear matrix. Theexpressionof certain transgenes(but not others) is strongly inhibited by linked prokaryoticsequences(2). Manyof the problems associated with transgenic mouseproduction wouldbe alleviated by the developmentof vectors that can be maintained

Annual Reviews

Annu. Rev. Immunol. 1988.6:25-48. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

TRANSGENIC MICE AND ONCOGENESIS

27

as autonomously replicating episomes.In principle, all injected eggs developing to term wouldbe transgenic, transmission to offspring wouldbe quantitative, problemsof insertional mutagenesisnonexistent, and most important, variation in expressionimposedby variable integration sites wouldbe absent. Attemptsto use bovine papillomavirus as an episomal vector have not been successful so far, however. In one study, many primarymice contained the vector in episomalform but weremosaic, and BPVDNA was apparently diluted further on subsequent breeding (7). two other studies, only integrated BPVDNA wasdetected (8). A promising advancehas been the serendipitous creation of autonomouslyreplicating elements containing defined segments of mousechromosomalDNAby injection of fertilized mouseeggs with a polyoma-based vector (9) together with trace levels of another plasmid(F. Cuzin, personal communication). Significantly, the incorporated mousesequences are homologousto conserved yeast centromeric sequences. Whetherthe propensity of the episomesto recombinationcan be overcomeremainsto be established. Retroviral

Vectors for Gene Delivery to the Germline

Considerableinterest exists in usingrecombinantretroviruses to introduce additional genes (including oncogenes)into the mousegermline. Amajor advantageof retroviral delivery is that it is technically less demanding, oncea suitable virus-producingline is available. Moreover,only a single gene is inserted per site, instead of a concatamer,whichmayadversely affect neighboringchromatinand transcriptional units (10). Severe problemsremain to be overcomewith the retroviral approach, however.To date, successful infection protocols use eight-cell stage embryos(11, 12)i hence, all founderanimalsare mosaic.Moreimportantly, however,expressionof the integrated provirus is usually suppressed,and this suppression is maintainedduring subsequentdevelopment(13). The mechanism of suppressionis not clear but mayinvolve methylation (14), inability of the long terminal repeat (LTR)to promotetranscription (1517), and/or transacting negativeregulatoryfactors (18). Twoapproaches have been taken to overcome suppression. One involved modifyingthe retroviral LTRby replacing the enhancerwith one knownto function within embryonalcarcinoma(EC) cell lines (12, 19). Theother involved redesign of the retrovirus to allow expressionof the geneof interest froman internal, cellular promoter(20-22). Despitepreliminarysuccesses,it is not yet clear howgenerallyusefulsuchvectorswill be. A potentially exciting use of recombinantretroviruses is to infect embryo-derived (ES) pluripotential cell lines capable of forminggermline chimeras(23). Since the viruses can be engineeredto express a selectable

Annual Reviews 28

CORY & ADAMS

markergeneas well as the geneof interest, infected EScells can be selected in vitro prior to either injection into blastocystsor aggregationwith eightcell embryos.Preliminaryexperimentsindicate that provirusesselected in .vitro for expressionin ECand EScells are stably expressedthroughout development and within~all tissues (22, 23). Amajor probleminherent this approach,however,is the tendencyof the ESlines to becomeaneuploid duringthe in vitro culture period and henceto lose their ability to form chimeras. Annu. Rev. Immunol. 1988.6:25-48. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Choice of Regulatory Sequences In attempting to target expression, a numberof different promoter/ enhancersequencescan be attached to the gene of interest (see 4 for detailed discussion). Theexpressionpattern of the engineeredhybridgene often conformsto that of the gene from whichthe regulatory region was derived. Novel patterns are sometimes observed, however, perhaps becauseof the unforeseen creation of a "recombinantenhancer," and/or the influence of flanking DNA. Thebest studied regulator for generalized expression is that of the metallothionein (MT)gene. The endogenous MTgene is expressed virtually all cells and is responsiveto several regulators, includingglucocorticoids and metals. Whilemost MTfusion genes have been expressed wellin liver, intestine, kidney,heart, pancreas,andtestes, unusualpatterns of expressionhavealso beenobserved(4). Threetissue-specific regulators havebeenparticularly successful: the immunoglobulin enhancersfor expressionin B lymphoidcells; the 5’ region of the rat elastase-I gene, for expressionin the exocrinepancreas;and the 5’ region of the rat insulin II gene, for expressionin the//cells of the endocrinepancreas. Theidentification of "strong" tissue-specific regulators for other cell lineages will greatly increase the versatility of the transgenic approach. ONCOGENES AND THE PROGRESSION MALIGNANCY

TO

Cancerhas long been perceived to be a multistage process. In molecular terms, this progressionis currently believedto involvethe stepwiseacquisition of activated oncogenes(24). Theexperimentalevidencesupporting this view derives froman analysis of the transforminggenesin DNA tumor viruses (25, 26) and fromin vitro transfection studies with primarycells, principally fibroblasts (27-29). Transformationwas foundto require the concertedaction of two genes. Significantly, effective collaboration was usually achievedonly if one of the pair of oncogenescamefrom the class

Annual Reviews

Annu. Rev. Immunol. 1988.6:25-48. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

TRANSGENIC MICE AND ONCOGENESIS

29

expressed in the nucleus and the other from the class expressed in the cytoplasm(24). Thecytoplasmicclass includes proteins related to growth factor receptors (erbB, fms), as well as proteins (e.g. ras) thought to regulate levels of critical second messengerssignalling interaction of growthfactors with their receptors. Thefunction of the nuclear class (e.g. myc, N-myc,fos) is unknownbut seemslikely to involve regulation of transcription. In markedcontrast to primaryfibroblasts, established but nontumorigenic lines such as NIH3T3can be readily transformed by a cytoplasmic oncogenewithout the mediation of a nuclear oncogene.This has led to the concept that the nuclear oncogeneprovides an "immortalization" function, while the cytoplasmiconcogenecompletesthe transformationprocess. Theconceptis an oversimplification, however,because transfection with a nuclear oncogenedoes not necessarily permit primary cells to growindefinitelyin vitro. Studieswith transgenicmicepermitdetailed investigationof the validity of the complementing oncogenemodelfor different cell lineages. Will a particular oncogeneprecipitate transformation as soon as expression is turned on, or does the disease progress in multiple stages? Are some transgenic strains essentially tumor-freeuntil crossed with a strain harboringa different oncogene?Cancells frompretumorigenictransgenic mice be readily grownas immortallines in vitro, or are they only able to do so if supplied with another activated oncogene?Are certain mousestrains moreprone than others to developingtumors from the action of a particular oncogene,and if so, do they harbor a susceptible proto-oncogene?

VIRAL ONCOGENES Introduction of a viral oncogeneinto the mousegermline opens up the possibility of testing its potential for transforming cell typesnot accessible to infection by the virus. Moreover,oncogenesisolated from viruses of other speciescan readily be investigated.

SV40 T Antigen Thefirst geneshownto exhibit oncogenicpotential in transgenic micewas the T antigen of the monkeypapovavirus SV40(30). The SV40virus transforms rodent and humancells in culture and induces tumors in hamsters but only rarely in mice. Newbornhamsters usually develop fibrosarcomasor, after intracerebral inoculation, choroid plexus papillomas, but adult animals can develop a wide range of tumors (30). The completetransformingpotential of SV40has been shownto be due to the large T antigen encodedby the early region. Since T antigen is expressed

Annual Reviews

Annu. Rev. Immunol. 1988.6:25-48. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

30

CORY & ADAMS

at the plasmamembraneas well as within the nucleus, it mayplay two different roles in the transformationprocess. Transgenicmice that carry the SV40early region linked to its natural regulator invariably developchoroid plexus tumorsat 3-5 monthsof age (30, 31). Thetumorsarise fromepithelial cells lining the ventricles of the brain. Tumordevelopmentdependson the presence of the SV40enhancer and the large T antigen codingsequencew~thinthe transgene, but not the small t antigen (31). Since most foundermicedevelopedthe samedisease, the chromosomallocation of the SV40antigen gene did not in general influencethe onset of disease (see below). T antigen wasreadily detected in tumorsbut not in unaffected tissues or in susceptible tissue prior to overt pathology.Withinthe tumors, the transgenic DNAwas usually amplified or rearranged. Nontumorigenic changes in SV40T antigen mice included thymic hypertrophyand kidney pathology;these tissues exhibited lowerlevels ofTantigen expressionthan did the choroid plexus tumors. It is not clear whetheramplified expression is sufficient to provoke tumorsor merelya prerequisite. Noris it knownwhychoroid plexustissue is permissiveto T antigen activation. TheSV40enhanceris thought to be inactivated during early development,and its reactivation in vivo maybe a rare event, dependentuponcell type. Reactivation mayreadily occur in vitro, however,becausemost apparently normaltissues from the SV40T antigen micereadily producedimmortalizedcell lines whenput into culture (30). Oneline of mice bearing an SV40T antigen gene remainedessentially free of tumors(32, 33). Significantly the level of expressionof T antigen in these micewas muchlowerthan in the other lines, both in vivo and in vitro (33), presumablydue to the chromosomal location of the transgene. Thecrucial check to tumor formation in this line may,however,be the immune response (32). Withthe low-incidencestrain, in whichonly 1 57 micedevelopeda choroidplexustumorwithin the first year of life, most mice mounta strong cytolytic T cell response after immunizationwith SV40virus. The developmentof cellular immunitypresumablyoccurs in response to chronic stimulation by the low levels of endogenousSV40T antigen expressedat the surface of cells. In markedcontrast, micewithin a strain with the normalhigh susceptibility to tumors failed to mount either a humoralor a cytotoxic response whenimmunized with virus, even though their response to vaccinia virus was normal. Suchstrains thus appearto be tolerant of T antigen, presumablybecauseantigen is expressed early in development.The high incidence of choroid plexus tumors thus appears to dependon the inability of mosttransgenic SV40T antigen mice to carry out immune surveillance.

Annual Reviews

TRANSGENIC ~tCE A~qDON¢OGENESIS31

Annu. Rev. Immunol. 1988.6:25-48. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

SV40 T Antigen Driven by Tissue-Specific Regulators TheSV40T antigen appears to be a potent tumorigenicagent for manycell types. Attachmentof a different regulatory sequenceavoids the negative regulation imposedby the S¥40 enhancer on transmission through the germlineand enablesexpressionto be directed to specific cell types. ~T/a" ANTIGEN Underthe control of the metallothionein enhancer, 13 of 16 primary transgenic mice exhibited a demyelinatingperipheral neuropathy associated with excessive numbersof Schwanncells (34). Myelination in the central nervoussystem wasapparently normal.Manyof the transgenic mice developedhepatocellular carcinomas(11 of 16) and islet cell adenomas (8 of 16), but only one exhibiteda choroidplexuspapilloma. Thosemice having a mild form of neuropathy and living long enoughto producelitters provedto mosaic.Their transgenic offspring all exhibited the severe peripheral neuropathy, all had hepatocellular carcinomasand 3 of 6 hadislet cell adenomas. ~t-CRYSTALLIN/T ANTIGEN Underthe

control of the murine ~-crystallin promoter/enhancer, transgenic SV40T antigen induced highly invasive lens tumors(35), eventhoughthis tissue has never beenobservedto become malignant under "natural" circumstances. T antigen expression and dysplasia wereobservedas early as midgestation, but it generally took 2 to 3 monthsfor invasive growthto occur outside the lens capsule. Whether the onset of malignancyrequired additional genetic changeis not clear. rNSULrN/T ANTIGEN Underthe control of the insulin regulatory region, S¥40T antigen transgene expression occurred exclusively in the fl pancreatic cells and culminatedin fl-cell tumors (36). On a normaldiet, transgenic mice frequently died suddenlyfrom hypoglycemia precipitated by hyperplasia of pancreatic fl cells. Thosespared by a high sugar diet succumbedlater (10-20 weeks)to pancreatic tumors. Presumablya second genetic alteration had occurred in such tumors, becauseno morethan 4 or 5 of the ~ 100 hyperplastic islets in a pancreas becameneoplastic. Lines developing "autoantibodies" against T antigen developed tumors considerablylater than did a line with no "autoantibodies." Again, this suggestsa role for immune surveillance (37). ELASTASE/T ANTIGEN Underthe

control of the elastase promoter region, SV40T antigen producedtumors of the exocrine pancreas (38). A marked hyperplasia wasevident in the pancreas within 17 days of gestation, but newbornmice retained pancreatic function and the DNAcontent of the cells wasdiploid. Onlyone monthlater, however,most cells had become tetraploid. The pancreas was soon riddled with nodules containing

Annual Reviews

32

CORY & ADAMS

Annu. Rev. Immunol. 1988.6:25-48. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

aneuploid cells, and the chromosome numbervaried betweennodules. The cells within these apparently independentclones were tumorigenicwhen transplanted. Theseresults suggesta role for SV40T antigen in the induction of karyotypicinstability via tetraploidization. Tumoronset maybe provoked by the subsequent loss of specific chr.omosome(s),perhaps becausecritical anti-oncogeneproducts are then either reducedin concentration or absent. T Antigen of Human Papovaviruses JCV and BKV The ubiquitous humanpapovaviruses JCV and BKVare structurally related to SV40.Themost divergent region of the three genomesis that encompassingthe region of replication and the enhancersequences, but homologybetween the T antigen genes is considerable (70-80%). The pathologyinducedby these viruses has beenreviewedby Smallet al (39). BKVreplicates preferentially in the kidney, producinga subclinical infection. Wheninjected into newbornhamsters, this virus causes brain tumors,insulinomas,and osteosarcomas. It is significant that in transgenic mice, the early region of BKVinducedrenal and liver tumors after 8-10 months(39). In humans,JCVhas beenlinked to the fatal demyelinatingdisease found in certain immunodepressed patients, someof whomalso develop glial tumors.Whilethe virus has not beenprovento cause these tumors, it does induce tumors in tissues of neural origin wheninjected into newborn hamsters. Significantly, transgenic mice mosaicfor the JCVearly region were found to develop metastasizing adrenal neuroblastomas(39). Those that were not mosaicdid not live long enoughto developtumors, because they developeda severe shaking disorder causedby dysmyelinationin the central nervoussystem(40). Thus, for both viruses, the pathologyinducedin transgenic mice by T antigen closely paralleled that thought to occur in immunosuppressed humansas a result of viral infection. Transgenicmice maythus provide excellent modelsfor humanvirus-induced disease. Bovine Papillomavirus Bovinepapillomavirus(BPV)(8) causes benignfibropapillomasin cattle. Ahigh proportionof infected animalsgo on to developtumorsif they graze on brackenfern, whichmayprovide a synergistic factor for oncogenesis. Certain other species, including C3HeB mice, developfibroblastic tumors wheninfected with the virus. Thegene(s) responsible for the transforming potential of the virus have not been fully characterized but are known from in vitro studies to reside within a fragmentcomprising69%of the genome.

Annual Reviews

Annu. Rev. Immunol. 1988.6:25-48. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

TRANSGENIC MICE AND ONCOGENESIS

33

Aline of transgenic micehas beenestablished from an animalinjected with a plasmidcarrying a partial tandemduplication of the completeBPV1 genome(8). Themice display a dramaticpropensity to developmultiple skin tumors, starting at about 8 monthsof age. Thetumors, whicharise frequently in areas prone to abrasion and wounding,are characterized by disorganizeddermallayers. In addition, the skin is abnormallythickened over wide areas. Bycontrast, 15 lines of transgenic mice harboring the BPV-69%-transforming region failed to develop tumors whenthe mice weremonitoredfor 10 months(Palmiter& Brinster, cited in 8), so the 31% region presumablycontributes an essential function for transformationin vivo. The long latency prior to transformation is interpreted to mean that additional genetic change(s)are required to precipitate oncogenesis. Extrachromosomal replication is probably a crucial factor because the tumors harbor BPVDNA in episomalform, as well as an amplified number of integrated copies. Theintegrated transgenewasunchanged in unaffected tissue. Studies like these with humanpapillomavirusDNA mightprovide valuable animalmodelsfor cervical cancer.

CYTOPLASMIC

CELLULAR

ONCOGENES

Ras The family of ras proto-oncogenesencodes membrane-bound cytoplasmic proteins believedto transducethe proliferative signals deliveredto the cell membrane by growthfactors and mitogens(for a recent review, see 41). Mutationof a c-ras gene at certain critical positions converts it into a potent transforminggene for NIH3T3 fibroblasts. Similarly mutatedgenes have been identified within manyhumanand animal tumors. Recent transgertic mousestudies have dramatically underlined the transforming potential of mutatedras genes. ~LASrASE/c-Ha-RAS Transgenicmice were producedcarrying either a normal or a mutated c-Ha-ras gene driven by the elastase promoter (42). Expressionof the transgenesprovedto be specific to acinar cells, but the level wasonly a fewfold higher than that of the endogenous Ha-rasgenes. Withthe unmutatedHa-rastransgene, no tumorsdeveloped.Nevertheless, anaplastic changeswereobservedin the pancreas of adult animals after about 11 months. Withthe mutatedgeneencodingvaline at position 12 instead of glycine, the results weredramatic. Theonly transgenic pupsto survive (5 of 19) were probably mosaic--the other 14 were dead or moribundat birth. The surviving mice all eventually developedpancreatic tumors. Whenanother

Annual Reviews

Annu. Rev. Immunol. 1988.6:25-48. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

34

CORY & ADAMS

20 transgenic micewereexaminedas fetuses, 18 had an abnormalpancreas. Dysplasiawasapparentas early as 14 days of gestation, the time expected for onset of elagtase-drivenexpression.Byday20, large-scale disruption of pancreatic architecture had ensued:Islets wereno longer recognizable, and insulin-containing cells werescattered throughoutthe cellular mass. Differentiation of the acinar cells appearedto be blocked.Whilethe pathology resembledthat of a carcinoma,the acinar cells maynot have been malignant:There wasno evidenceof aneuploidy,invasiveness, or metastasis, and the cells did not initiate tumors in syngeneic or nude mice. Thus, the disease maymore properly be regarded as a lethal polyclonal proliferation than a malignancy. If the mosaicelastase/ras micecontainedevena fewtransgenic cells in the pancreas, howdid they survive for so long without developing a pancreatic carcinoma? One obvious possibility is that the level of expression mayhave been lower. Anotherpossibility is that ras has a muchmoreprofoundeffect in embryonicthan adult cells. A third, very intriguing, suggestion (42) is that the transformedphenotypewas suppressedin the presenceof an excessof normalcells, as postulated by Land et al (29) for fibroblast transformation. WAP/c-Ha-RAS TOinvestigate the consequencesof expressionof a ras oncogene in mammary epithelial cells in response to lactogenic hormones, transgenic lines were constructed bearing a mutatedHa-rasgene driven by the promoterregion of the gene encodingmurinewheyacidic protein (Wap)(43). Twolines exhibited no expression, but two others derived from female founders showedtissue-specific expression. A low level of Wap-rasRNAwas detected in the mammary glands (and brain) of lactating females, and no expression could be detected after lactation terminated.Maleswithin these lineages did not express the transgene. After 11 months, one lactating founder female developedmammary tumors that expressed elevated levels of Wap-rasRNA. In one unusual line, expression was obtained in the salivary gland of males, presumablydue to the unusual location of the transgene (the chromosome).Five animals developed adenocarcinomasof that gland, but only after 9 months; this suggests the need for additional genetic change. Again, Wap-RasRNAlevels were higher in the tumors than in nonmalignantsalivary tissue from the sameanimals. The transgene was neither amplifiednor rearrangedin the tumors,and its transcription rate was unchanged.Increased expression maythus have beendue to an alteration in mRNA stability. MMTV/v-Ha-RAS Thirteen strains of mice wereproducedcarrying the v-Ha-

Annual Reviews

Annu. Rev. Immunol. 1988.6:25-48. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

TRANSGENIC MICE AND ONCOGENESIS

35

ras oncogene under the control of the mousemammarytumor virus (MMTV) promoter (78). As expected, the transgene was predominantly expressedin the mammary tissue and in the salivary glands, with variable expression in spleen, thymus, lung, seminal vesicle and the Harderian gland. Expression in the Harderian gland provokedmassive though nonmalignant hyperplasia, resulting in severe secondary bilateral exophthalmia. Most (10) strains developed adenocarcinomasof the mammary and salivary glands and, occasionally, lymphomas. The stochastic nature of tumor onset and the apparent monoclonality of the tumors strongly suggestedthat additional genetic changeshad occurred prior to the onset of malignancy. In summary,a transgenic mutant ras gene can provokeabnormallevels of proliferation in several different cell types. Theseverity of disease apparently dependson the level of expressionand cell type. Theonset of true neoplasia usually (perhapsalways)requires additional change. Transgenic Hernopoietic

Growth Factor Gene

Theproliferation, differentiation, and functionalactivation of the myeloid hemopoieticcell lineagesis controlled by a groupof glycoproteinsreferred to as colonystimulating factors (CSFs). The granulocyte/macrophage CSF (GM-CSF) stimulates proliferation and differentiation of granulocytes, macrophages,and eosinophils (44). Autocrinestimulation of factor-dependent but nontumorigenicimmortalizedcell lines renders themmalignant (45-47).Thus,it is of the greatest interest to ask whethertransgenicmice programmedfor constitutive CSFproduction are predisposed to tumor development. Twotransgenic mouselines were generated that contained the murine GM-CSF gene expressed from a retroviral LTR(48). The transgenic mice had elevated levels of GM-CSF in the serum, urine, peritoneal cavity, and eye. Striking pathogeniceffects ensued. Theeyes of newborntransgenic pups were opaque, due to an abnormalaccumulationof activated macrophages;the associated retinal damageled to blindness. Theperitoneal and pleural cavities accumulatedactivated macrophages in large numbers.The mice died at 2-4 months, and a high percentage exhibited progressive muscle wasting. Histological examination revealed macrophage-containing lesions in their striated muscletissue. Expressionof the transgene wasdetected in the peritoneal cell populationand in striated muscleand eye tissue harboring macrophagelesions. No tumors have yet been detected, but the severity of disease maynot allow sufficient time for the accumulationof additional genetic change(s).

Annual Reviews 36

CORY

Annu. Rev. Immunol. 1988.6:25-48. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

NUCLEAR

& ADAMS

CELLULAR

ONCOGENES

Fos The c-los proto-oncogene encodes a nuclear polypeptide implicated in the control of cell proliferation. One of the earliest responses of manycell types to growth factor or mitogenstimulation is a transient burst of c-fos synthesis. The gene mayhave other roles, however, since high constitutive levels have been found in extra-embryonic tissues and in macrophages(for a review, see 49). The gene was first identified as a viral oncogene. Two independent isolates of murine osteosarcoma viruses carry sequences homologous to c-fos: FBRmurine sarcoma virus, in which the transforming protein differs from c-fos mainly by a frameshift mutation at the C-terminus, and the more aggressive FBJ-murine sarcoma virus, which encodes a greatly altered version of the protein. Both viruses induce chondro-osseoussarcomas when injected into newborn mice. A mutation within the evolutionarily conserved central portion is necessary for immortalization, while COOHterminal changesare critical to their transforming properties (50). Transgenic mice have been produced bearing a murine c-fos gene controlled by the humanmetallothionein promoter/enhancer (51). Twoconstructs were used. Onehad the natural 3’ untranslated region of the c-fos gene, which contains a mRNA-destabilizing sequence. This region was replaced in the second construct by the 3’ LTRfrom the FBJ virus. In eight transgenic strains involving the natural 3’ end, very little expression could be detected, except after treatment with cycloheximide, which is thought to inhibit degradation of c-fos mRNA. In contrast, two of the five transgenic lines carrying the 3’ LTR-modifiedgene exhibited high levels of expression in pancreas, kidney, brain, heart, muscle, and lower levels in lung and salivary gland. Liver expression had been expected but was absent. While none of the animals developed tumors within the first 8 months, a marked abnormality in bone formation became apparent soon after birth. Swollen tibiae were evident in some2-3 weekold mice. Bonelesions were subsequently found in other mice by X-ray analysis. The swellings did not increase after 4 weeks of age. Histological analysis revealed a disturbance in the normal process involved in bone remodelling, with much more bone formation than bone resorption. The level of c-fos expression probably determines the severity of the disease, because a c-fos gene linked b promoter induced multiple swellings in all leg bones, not just to the H-2K the tibiae. These results point to a significant role for the normalfos gene in regulating bone morphogenesis. In the MT/c-fos mice, none of the other tissues expressing the transgene

Annual Reviews

Annu. Rev. Immunol. 1988.6:25-48. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

TRANSGENIC MICE AND ONCOGENESIS

37

exhibited detectable abnormalities. However,H-2/c-fos miceexhibit perturbations of lymphopoiesis (E. Wagner,personal communication), quantitative effects maybe involved.ThethymicT cells exhibiteda marked depressionin the proportionof cells bearingboth L3T4and Lyt-2 markers. Dueto a defect in T cell maturation, these mice are immunologically incompetent. Thevery early onset of the bonelesions inducedby the c-fos transgenes, togetherwith their intriguing similarity to the frank osteosarcomas induced by v-fos bearingretroviruses, suggeststhat the lesions can be regardedas a hyperplastic state from which tumors could arise should additional genetic changetake place. Indeed,tumorshavearisen in someof the older c-fos mice. The ready induction of tumors by the retroviruses probably reflects the structural differences betweenv-fos and c-fos and/or a higher expressionlevel. Myc The c-mye proto-oncogeneis a nuclear phosphoproteinwith DNA binding activity. Its function remainsunclear but almostcertainly involves regulation of cell proliferation (52). Arapid increasein my¢expressionoccurs whenresting (Go)cells of several lineages are stimulatedto enter the cell cycle by mitogensor growthfactors. Recentevidencesuggests that c-myc protein plays a role in DNA synthesis (53). The fundamentalmechanism releasing the oncogenicpotential of c-myeis believed to be deregulation of its expression (54). Nochangein the sequenceof the polypeptide required. Totest the hypothesisthat deregulatedc-mycexpressionpredisposesto neoplasia, several strains of transgenic mice have been developedthat carry c-myelinked to different regulators (55, 56). Micebearinga normal mycgenetogether with several kilobases of 5’ and 3’ flanking DNA failed to developtumors(56). In contrast, those in whichmycexpressionhad been subjugated to a strong heterologouscontrol region exhibited a dramatic predisposition to tumors (55-57). Diverse tumor types were provoked, dependingon whichtissues expressedthe deregulatedgene. Beforereviewing these malignanciesin detail, however,it is worthnoting that a c-rnyc oncogenemaynot provoketransformation of all cell types. Thus, tumors did not developin lung, pancreas,and salivary gland expressingrelatively high levels of a deregulatedmy¢gene(42, 57). MMa’V/r,~YC Several strains of mice were developedthat bear a humancmyegene linked to the LTRof the mousemammary tumor virus (MMTV), whichprovides a hormonallyinducible promoter (55). The tissue distribution of expression varied betweenthe lines, presumablydue to the

Annual Reviews

Annu. Rev. Immunol. 1988.6:25-48. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

38

CORY & ADAMS

influence of the chromosomal insertion site. In mostlines, expressionwas restricted to the breast, salivary gland, and occasionallythe testis. Two multiparous founder animals developed malignant adenocarcinomasof the breast. The transgene was not amplified in the tumors but was expressed at higher levels than the endogenousc-mycgenes. In the strain studied in detail, all femaletransgenic offspring developedbreast tumors during their second or third pregnancy. Thus, mycexpression stimulated lactogenically appears to predisposethe mice to the onset of mammary adenocarcinomas. Not all the mammary glands in an individual becamemalignant;this strongly suggests that additional genetic changeis required(55). One exceptional MTV-myc lineage proved to express the transgene in diverse tissues, includingtestis, pancreas,lung, spleen, liver, and(in low amounts)kidney. This strain allowedan assessmentof the range of tissues susceptible to transformation involving c-mye (57). Some50%of the animals developedtumors, with a meanlatency of 14 months. Thevariety wasstriking: testicular tumors, B and T lymphomas, and mastcell tumors, as well as breast adenocarcinomas.Mice carrying a murine c-myc gene coupledto the SV40promoter/enhanceralso developeda range of tumors: a lymphosarcoma,a renal carcinoma, and a fibrosarcoma (56). Clearly deregulatedexpressionpredisposesdiverse cell types to becomemalignant. IG ENHANCER/MYCThe strong

association between c-myc deregulation and lymphoidneoplasia (54) provided a compellingcase for using transgenic mice to test the consequencesof constitutive mycexpressionwithin the lymphoidlineage. Twotransgenes were utilized, one involving the immunoglobulinheavy chain enhancer (E/~) and the other the kappa enhancer (Ex) (56). E#-myc geneused had been isola ted from an unusual plasmacytomain which 2.3 kb of the E# enhancer region had inserted just upstreamfrom the c-myc promoters (58). The Ex-mycgene wasa synthetic construct in whichmycexpression wascontrolled by the x light chain enhancerligated to the SV40promoter.TheE#-myctransgene proved remarkably potent: At least 13 of 15 primary mice developed lymphomas within a few monthsof birth. Thus, the heavy chain enhancer apparently dominatesexpression in manychromosomal contexts. The Exmyctransgene also induced lymphomas, albeit with lower frequency(6 of 17 primarymice) and longer latency. The lymphoidspecificity wasabsolute; no other tumor types were found either in the original study or subsequently. The high predisposition to tumor developmentin E#-mycmice has proven highly heritable. The mice typically succumbto a disseminated lymphoma affecting most lymphnodes, the spleen, and often the thymus;

Annual Reviews

Annu. Rev. Immunol. 1988.6:25-48. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

TRANSGENIC MICE AND ONCOGENESIS

39

the disease is accompaniedby leukemia. All tumors so far analyzed (> 50) have proven to be of B lymphoid origin, even those primarily involving the thymus. All exhibit B lineage surface markers and immunoglobulin gene rearrangements; none carry Thy-1; and only one or two have undergone rearrangement of the a or fl T cell receptor loci. The absence of T cell tumors was initially surprising, since a complete immunoglobulin# transgene is expressed in T as well as B cells (59). But no expression of the Et~-mycgene has been detected in T cells (see below). A minority of the E#-rnyc tumors are comprised of mature B cells expressing sIg, while the rest can be assigned to different pre-B stages, exhibiting either D-J or V-D-J heavy chain rearrangement. None so far studied represents the very early maturation stage that lacks any heavy chain rearrangement. Most are relatively easy to establish as permanent lines in culture, and manyof the pre-B cell lines maturein vitro to sIg + B cell lines (A. Harris, W. Langdon, S. Cory, unpublished). The rearrangement patterns in certain mice suggested that maturation also sometimes occurs in vivo. Thus, myc-induced tumorigenesis need not prevent some subsequent differentiation. Compared to B lineage tumors generated by retroviruses bearing a variety of other oncogenes,including v-abl, a significant difference in the phenotype of El~-myc tumors is the absence of the 6C3 surface marker (W. Langdon, unpublished). This antigen (60) is also expressed at levels on stromal cells that support pre-Bcell proliferation and is speculated to be a growth factor or receptor (61). Tumors produced by myc retrovirus also lack this marker (62). Thus, it seems likely that myc-induced tumorigenesis involves a pathway different from that imposed by other oncogenes. Another distinctive feature of most E#-myctumors is the lack of the Lyl marker (W. Langdon, unpublished). This antigen is present on the surface of most conventional B lymphoid tumors and also on nontumorigenic B cell lines derived from normal mice (63). Most conventional lines therefore apparently derive from the minor, seeminglydistinct, Ly 1 + subset of B cells (64), whereas El~-mye-mediatedtransformation may garner a morerepresentative sample of all B lineage cells. To date, no plasmacytomas have arisen spontaneously in E#-mye mice, despite the fact that conventional plasmacytomas all carry chromosome translocations produced by recombination of the c-myc locus with an Ig heavy chain locus (54). This paradoxical observation maysimply reflect the different genetic background; the E#-myc gene is on a C57BL/6× SJL/J background, whereas plasmacytomas usually arise in BALB/eor NZB mice. Nevertheless, it maybe possible to induce plasmacytomasin E#-rnyc mice by providing the appropriate milieu. Experiments are currently in

Annual Reviews 40

CORY& ADAMS

Annu. Rev. Immunol. 1988.6:25-48. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

progress (A. Harris, unpublished results) to determine whether pristane injection, which induces granulomas, will promote plasmacytoma formation in El~-myc mice as it does in BALB/cmice. E/z-myc induced tumorigenesis involves additional genetic change Despite the almost invariant onset of tumors in E#-mycmice, several observations argue that deregulated myc expression is almost certainly not the only critical factor. First, the lymphoidtissues of young E#-mycmice lacking any overt sign of disease express the transgene at the samelevel as do E#myc tumor cells (65). Nevertheless, in contrast to the tumor cells, they do not provoke malignancy on transplantation (66). Second, while the transgene disturbs B cell development in all E#-myc mice, even before birth (66), lymphomasarise sporadically--typically between one and six months of age (56, 67). Third, the pattern of immunoglobulin gene rearrangement indicates that nearly all the lymphomasare monoclonal (56). Finally, and most definitively, DNAfrom several of the El~-myc tumors transforms NIH3T3fibroblasts (S. Cory, W. Alexander, J. Adams, unpublished results). Since the EIz-myc gene does not transform NIH3T3 cells, these tumors must harbor an additional activated oncogene, presumably one that can collaborate with c-myc. The nature of the transforming gene(s) is presently under study. While all E#-myc mice eventually develop tumors, a minority succumb faster than most, and another group lag conspicuously. It seems possible that the variation in rate of onset reflects the variable genetic background of the mice. Because transgenic mice are produced more efficiently from hybrid eggs (2), each founder was an F2 derived from the C57BL/6and SJL/J strains, and the major colony has been maintained as an F2 pool. Preliminary backcross experiments (A. Harris, unpublished results) have revealed a major influence of strain: E#-myc mice of a largely C57BL/6 backgroundall still develop tumors, but the rate of onset is significantly lower. It appears relevant that old SJL mice can develop a lymphoid malignancy,perhaps as a result of a single recessive trait (68). The pre-neoplastic state in E#-mycmice To investigate howconstitutive c-myc expression establishes a high predisposition to malignancy, we analyzed B cell lymphoid differentiation in prelymphomagenic EIz-myc mice (66). The most conspicuous change was a remarkable polyclonal expansion of pre-B cells, somewhatat the expense of mature B cells. By 18 days of gestation, the proportion of pre-B cells in the fetal liver was already twice that in normal littermates. The number of pre-B cells increased dramatically after birth, reaching a plateau of about 65%of the bone marrowcells at 3 to 4 weeks of age. Pre-B cells were also found in the spleen; this is not the case in normal mice. The cells mayhave been

Annual Reviews

Annu. Rev. Immunol. 1988.6:25-48. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

TRANSGENIC MICE AND ONCOGENESIS

41

generatedin situ as a result of increased splenic hemopoiesis.It is significant, however, that pre-B cells were not detected in the lymph nodes or thymus, even though these are the most commonsites for tumors. Thus, acquisition of the ability to invade the lymph nodes and thymus may be a critical step in progression towards malignancy. No significant changes were detected within the thymus of prelymphomagenicEl~-myc mice: The proportions of the four major T cell populations were unaltered. Because of reduced numbers of sIg + B cells, the proportion of T cells in the lymph node was greater than normal. The size of these cells was normal, however. Both the pre-B and B cells in the El~-mycmice were notably larger than most equivalent cells in normal mice. Late pre-B cells are thought to enter a quiescent nonproliferative phase as they mature to B cells. In El~-myc mice, however,no small pre-B or B cells were detectable. In addition, more than twice as manyof the pre-B and B cells were actively cycling in E#myc than in conventional mice. Since the analysis did not distinguish betweennoncycling cells and those traversing G1, it is conceivable that as a result of constitutive c-myc expression, none of the cells ceased cycling prior to their death. A large proportion of the cycling cells must in fact die to account for the steady state level found in adult El~-mye mice. The factors regulating the plateau level are not known,but one obvious possibility is that the supply of the requisite growthfactor(s) is limited. Overall, the total numberof pre-B cells in El~-myc mice was elevated over 4-fold, while the numberof mature sIg + B cells was depressed about 30% (66). Moreover, fewer splenic B cells had matured to surface IgD expression. These observations raised intriguing questions regarding the immunologicalcompetenceof B cells continually subjected to constitutive myc expression. Et~-mycB cell function was therefore investigated both in vitro and in vivo (68). While in vitro stimulation with either mitogens antigens led to proliferation and antibody production, the frequency of responsive B cells in E~-myccultures was only 30%that of B cells from control cultures. In vivo responses were sometimesdelayed but eventually reached normal levels, and switching from IgM to IgG production occurred. Thus, deregulated myc expression appears to retard but not prevent B cell differentiation; consequently, immunologicalfunction is not grossly impaired. To account both for the increased numberof cells early in the B lineage in Et~-mycmice and for the maintenance of at least a high proportion of the cells in cycle, it was proposed that c-myc normally plays an important role in control of differentiation as well as mitogenesis (66). The level c-myc expression was envisaged to set the balance between self-renewal and maturation within a cell lineage, the maturation requiring decreased

Annual Reviews

Annu. Rev. Immunol. 1988.6:25-48. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

42

CORY & ADAMS

levels. Constitutive expressionwouldthus favor self-renewal. Additional evidencesupportingthis modelis that deregulated mycexpressionwithin mouseerythroleukemiacells blocks the terminal differentiation induced by dimethylsulfoxide (69, 70). The model maynot hold for all lineages, however, because differentiated keratinocytes maintain high myc expression(71). All changesnoted in the E#-mycmice reflect alterations within the B lymphoidcompartment,consistent with evidence that the transgene is apparentlynot expressedin other lineages (65). It is significant that the level of transgene expression was found to be comparableto that in activated normallymphocytes.Thus, constitutive expression at physiological levels of a normalcellular genecan elicit all the profoundbiological effects cited above. This conclusion dramatically underlines the importance of understanding the molecular mechanismthat normally regulates c-myc expression. Astriking feature of all E#-mycB lymphoidcells, whetheror not they have becometumorigenic,is that the endogenousmycalleles are transcriptionally silent (56, 65), as is the untranslocatedmycallele in plasmacytomasand Burkitt lymphomas (72, 73). These results strongly support a negative feedbackmodelfor control of mycexpression(74, 75). A myc polypeptide concentration above a threshold level (which may vary betweenlineages or differentiation stages) is hypothesizedto repress further myctranscription, either directly or indirectly. TheE#-myctransgene, like a translocated mycallele, presumablyis recalcitrant to this repression. In viewof the immortalizingrole ascribed to the c-rnyc oncogene,it was surprising to find that pre-neoplasticE#-mycB lineage cells wereno easier to growin culture than their normalcounterparts. Whencultured under conventional conditions, with no source of growth factors other than serum, E#-myepre-B marrowcells in fact died more rapidly than their normal counterparts (W. Langdon, unpublished results). Moreover, splenic B cells from E#-mycmice did not display unlimited proliferative capacity, even in the presence of mitogens and rich sources of growth factors (68). Thus, the description of my¢as an immortalizinggene may be an oversimplification. At present, E#-mycpre-B cells can only be maintainedin vitro within Whitlock-Wittecultures (W. Langdon,unpublished results), in which unidentified growth factors are provided by an adherent stromal layer grownfrom normal bone marrowcells. In such cultures, the transgenic cells were again found to be larger than normalpre-B cells in parallel cultures. At least twiceas manywerein cycle, but theygrewto cell densities only slightly higher than those of their normalcounterparts, presumably

Annual Reviews

Annu. Rev. Immunol. 1988.6:25-48. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

TRANSGENIC MICE AND ONCOGENESIS

43

because growth factor levels were limiting. Whenisolated from bone marrow,manyEla-myc pre-B cells display Ia (66), a marker normally confined to more mature cells within the B lineage. After one weekin culture, however,Ia expressionwaslost, perhapsbecausethe cultures lack factor(s) essential for Ia expression.Thus, all evidenceto date suggests that B lymphoidcells constitutively expressing c-myc maintain their requirement for growth factors, although perhaps at somewhatreduced levels (see also 76). Theseresults contrast markedlywith the report that infection with a v-myccontaining retrovirus abrogates the factor dependenceof certain hemopoietic cell lines (77). It is of great interest to see whetherthe entire E~-myctumorigenic processcan be reconstructedin vitro. Thecells in several long-termcultures of transgenic marrowcells markedlychangedtheir growthcharacteristics after several monthsof growth(W.Langdon,unpublished), reaching 10fold higher saturation densities but still requiting the stromallayer. One such culture finally becamefactor independentand tumorigenic.Thus, the events required in vivo for onset of tumors maywell include mutations subvertingfactor requirements. In summary,analysis of prelymphomagenic E#-mycmice has provided someinsight into whydysregulated mycexpression creates tumor-prone mice. Whenimposed on the B lymphoid lineage, constitutive myc expressioncauses a 4-fold elevation in cell numbers,and morethan twice as manycells are replicating. Since the total numberof divisions has been increased by an order of magnitude,the risk of genetic accident is also significantly increased. Similar conclusions presumablyhold for other lineages expressing a deregulated myc gene. The immunoglobulin rearrangement mechanismcould be an added risk factor for lymphoid cells if, as in E#-mycmice, the expandedpopulationis primarily composed of cells actively undergoingDNA rearrangement.These factors maynot, however,fully account for the very high predisposition to tumoronset, and it maywell provethat mycacts moredirectly to increase the frequency of mutation. ONCOGENE

COOPERATIVITY

Theconclusion frommost of the transgenic oncogenestudies discussed is that expressionof one oncogeneis insufficient for malignancyto develop. (Ras oncogeneexpressionin pancreatic acinar cells maybe an exception-see 42.) Thetransgenic miceoffer uniqueopportunitiesto investigate the consequenceof exposingthe relevant cells to the action of additional oncogenes.Oneobvious wayis via retroviral infection. Neonatal mice carrying one oncogeneas a transgene can be infected with a virus bearing

Annual Reviews

Annu. Rev. Immunol. 1988.6:25-48. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

44

CORY & ADAMS

the secondoncogeneand monitoredfor tumoronset. Alternatively, cells isolated fromthe transgenic mice can be infected in vitro and their subsequent growthproperties monitoredin vitro and in vivo. Studies initiated along these lines with pre-B cells from E#-mycmice are proving most informative (W. Alexander, J. Adams,W. Langdon,S. Cory, unpublished results). Whenneonatal EIz-mycmice wereinfected with a helper virusfree virus carrying a mutated Ha-ras gene, lymphomainduction was greatly accelerated. Analternative strategy to investigate cooperativityis to cross lines of transgenic mice that express different oncogeneswithin the sametissue. Myc/rascooperativity has been demonstratedin breast epithelial cells by breeding a strain of MMTV-ras transgenic mice with an MMTV-myc line (78). The latency for onset of mammary carcinomas was considerably reducedbut their time of onset wasstill variable and the tumorsdid not involve the entire breast. Thusadditional somatic events appearedto be required for the onset of malignancyeven in the presence of both a deregulated mycgene and an activated ras gene. CONCLUSIONS

AND FUTURE

PROSPECTS

It is alreadyclear that transgenicmicewill substantially deepenour understanding of oncogeneaction. The efficacy of several genes as tumorpromotingagents in the living animalhas been demonstratedconclusively. In general, activity of morethan one cancer-promotinggene seemsto be required for the emergenceof a tumorigenic clone. The myc and ras oncogeneshave been shownto collaborate in the transformation of two cell types. Wecan look forwardto seeing the results of a great variety of such experiments, and these should establish the "rules" for oncogene cooperativityin diversecell lineages. Theclear demonstrationof an altered but preneoplastic phase in the transgenic animalsis particularly important. Duringthis stage, the biological effect of the primaryoncogenecan be analyzed.Moreover,attempts can be madeto alter the courseof the disease and perhapsevento prevent the onset of malignancy.Just as important,the effect of a rangeof chemical agents can be investigated for their tumor-promoting activity. Thecharacterization of additional tissue-specific promoter/enhancer sequenceswill extend the versatility of experimentstargeting oncogene expressionto specific cell lineages. Conversely,it will be of considerable interest to comparethe biological effects of different oncogeneswithin the same cell type. Already a comparisonhas been madeof the effects of mycand ras within mammary epithelial cells (55, 78). Moreover,several different oncogeneshave been coupledto the immunoglobulin heavychain

Annual Reviews

Annu. Rev. Immunol. 1988.6:25-48. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

TRANSGENIC MICE AND ONCOGENESIS

45

enhancer (S. Cory, A. Harris, J. Adams,unpublished results), including N-myc, N-ras, v-myb, v-abl, and bcr-v-abl, a construct mimicking the hybrid oncogenethought to play a central role in chronic myeloid leukemia. Most of these genes have provoked lymphoid neoplasia in transgenic mice. The tumors observed to date include plasmacytomas and T lymphomas, as well as pre-B and B lymphomas. Transgenic mice harboring different growth factor genes will be an area for fruitful investigation, irrespective of whether their deregulated expression provokes tumors. In fact, certain oncogenes maywell prove to be growth factors expressed at very specific times of development: Int-2 has already been implicated as an embryonicgrowth factor (79). Finally, transgenic mice mayprove crucial to analysis of the recessive oncogenes, genes encoding products that normally prevent the emergence of tumors. High-level expression of an antisense copy of such genes in tumorigenic mice might mimic their loss and thereby induce malignancy. Since a strong candidate for the recessive retinoblastoma (Rb) susceptibility gene has been cloned recently (80), anti-Rb transgenic mice may well be the first of such analyses attempted.

Literature Cited 1. Gordon, J. W., Ruddle, F. H. 1983. Gene transfer into mouseembryos: Production of transgenic mice by pronuclear injection. Methods Enzymol. 101: 41133 2. Brinster, R. L., Chen, H. Y., Trum¯ bauer, M. E., Yagle, M. K., Palmiter, R. D. 1985. Factors affecting the efficiency of introducing foreign DNAinto mice by microinjecting eggs. Proc. Natl. Acad. Sci. USA 82:4438-42 3. Brinster, R. L., Palmiter, R. D. 1986. Introduction of genes into the germ line of animals. The Harvey Lectures 80:1-38 4. Palmiter, R. D., Brinster, R. L. 1986. Germline transformation of mice. Ann. Rev. Genet. 20:465-99 5. Hogan, B., Costantini, F., Lacey, E. 1986. Manipulatin9 the mouse embryo: A laboratory manual. Cold Spring Harbor Lab., Cold Spring Harbor, Me. 6. Woychik, R. P., Stewart, T. A., Davis, L., D’Eustachio, P., Leder, P. 1985. An inherited limb deformity created by insertional mutagenesis in a transgenic mouse. Nature 318:36-41 7. Elbrecht, A., DeMayo,F. J., Tsai, M. J., O’Malley, B. W. 1987. Episomal maintenance of a bovine papilloma virus vector in transgenic mice. Mol. Cell. Biol. 7:1276-79

8. Lacey, M., Alpert, S., Hanahan, D. 1986. Bovine papillomavirus genome elicits skin tumors in transgenic mice. Nature 322:609-12 9. Rassoulzadegan, M., L6opold, P., Vailly, J., Cuzin, F. 1986. Germline transmission of autonomous genetic elements in transgenic mousestrains. Cell46:513-19 10. Wagner,E. F., Covarrubias, L., Stewart, T. A., Mintz, B. 1983. Prenatal lethalities in mice homozygous for human growth hormone gene sequences integrated in the germ line. Cell 35:647-55 11. Jaenisch, R. 1976. Germline integration and Mendelian transmission of the exogenous Moloney leukemia virus. Proc. Natl. Acad. Sci. USA73:1260-64 12. Van der Putten, H., Botteri, F., Miller, A. D., Rosenfeld, M., Fan, H., Evans, R., Verma, I. 1985. Efficient insertion of genes into the mouse germ line via retroviral vectors. Proc. Natl. Acad. Sci. USA 82:6148-52 13. Jaenisch, R., J/ihner, D. 1984. Methylation, expression and chromosomal position of genes in mammals. Biochem. Biophys. Acta 782:1-9 14. Stewart, C. L., Stuhlmann, H., J/ihner, D., Jaenisch, R. 1982. De novo methylation, expressionand infectivity of retro-

Annual Reviews

Annu. Rev. Immunol. 1988.6:25-48. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

46

CORY & ADAMS

viral genomesintroduced into embryonal carcinomacells. Proc.Natl. Acad. Sci. USA79:4098-4102 15. Gautsch, J. W., Wilson, M. C. 1983. Delayedde novo methylationin teratocarcinomasuggests additional tissuespecific mechanisms for controlling gene expression. Nature301:32-37 16. Niwa,O., Yokota,Y., Ishida, H., Sugahara, T. 1983. Independentmechanisms involved in suppression of the Moloney leukemia virus genomeduring differentiation of murine teratocarcinoma cells. Cell 32:1105-13 17. Linney, E., Davis, B., Overhauser,J., Chao, E., Fan, H. 1984. Nonfunction of a Moloneymurine leukaemia virus regulatory sequence in F9 embryonal carcinomacells. Nature308:470-72 18. Gorman,C. M., Rigby, P. W.J., Lane, D. R. 1985. Negativeregulation of viral enhancersin undifferentiated embryonic stem cells. Cel142:519-26 19. Franz, T., Hilberg, F., Selinger, B., Stocking, C., Ostertag, W.1986. Retroviral mutantsefficiently expressedin embryonalcarcinomaceils. Proc. Natl. Acad. Sci. USA83:3292-96 20. Soriano, P., Cone, R., Mulligan, R., Jaenisch, R. 1987. Tissue-specific and ectopic expression of genes introduced into transgenicmiceby retroviruses.Science 234:1409-13 21. Rubenstein,J. L. R., Nicolas, J. F., Jacob, F. 1984.Constructionof a retrovirus capable of transducing and expressing genes in multipotential embryonic cells. Proc. Natl. Acad.Sci. USA 81:7137~,0 22. Stewart, L., Vanek,M., Wagner,E. F. 1985. Expressionof foreign genes from retroviral vectors in mouse teratocarcinomachimaeras. EMBO J. 4: 37019 23. Robertson,E., Bradley, A., Kuehn,M., Evans, M. 1986. Mousegermline transmission of gene sequences introduced into cultured pluripotential cells by a retroviral vector. Nature323:445~,8 24. Weinberg, R. A. 1985. The action of oncogenesin the cytoplasmand nucleus. Science 230:770-76 25. Rassoulzadegan,M., Cowie,A., Carr, A., Glaichenhaus, N., Kamen, R., Cuzin,F. 1982. Theroles of individual polyomavirus early proteins in oncogenic transformation. Nature300: 71318 26. Vander Elsen, P., de Pater, S., Houweling, A., vander Veer,J., vander Eb, A. 1982. The relationship betweenregion Ela and Elb of humanadenoviruses in cell transformation. Gene18:175-85

27. Land, H., Parada, L., Weinberg,R. A. 1983. Tumorigenicconversion of primaryembryofibroblasts requires at least two co-operating oncogenes. Nature 304:596~00 28. Ruley, E. 1983. Adenovirusearly region 1Aenables viral and cellular transforminggenesto transformprimarycells in culture. Nature304:602~5 29. Land,H., Chen,A. C., Morgenstern,J. P., Parada,L. F., Weinberg,R. A. 1986. Behaviourof mycand ras oncogenesin transformation of rat embryofibroblasts. Mol.Cell. Biol. 6:1917-25 30. Brinster, R. L., Chert, H. Y., Messing, A., Van Dyke, T., Levine, A. J., Palmiter, R. D. 1984. Transgenicmice harboring SV40T-antigengenes develop characteristic brain tumors. Cell 37: 367-79 31. Palmiter, R. D., Chen,H. Y., Messing, A., Brinster, R. L. 1985. SV40enhancer and large-T antigen are instrumentalin developmentof choroid plexus tumors in transgenic mice. Nature 316:45740 32. Faas, S., Pan, S., Pinkert, C., Brinster, R., Knowles, B. 1987. Simian virus 40 SV40-transgenic mice that develop tumoursare specifically tolerant to SV40 T antigen. J. Exp. Med.165:417-27 33. VanDyke,T., Finlay, C., Levine, A. J. 1985. A comparisonof several lines of transgenic mice containing the SV40 early genes. Cold Spring HarborSymp. Quant.Biol. 50:671-78 34. Messing,A., Chen, H. Y., Palmiter, R. D., Brinster, R. L. 1985. Peripheral neuropathies, hepatocellular carcinomasandislet cell adenomas in transgenic mice. Nature 316:461~3 35. Mahon, K. A., Chepelinsky, A. B., Khillian, J. S., Overbeek,P. A., Piatigorsky, J., Westphal, H. 1987. Oncogenesis of the lens in transgenic mice. Science 235:1622-28 36. Hanahan,D. 1985. Heritable formation of pancreatic r-cell tumoursin transgenic miceexpressing recombinantinsulin/simian virus 40 oncogenes. Nature 315:115-22 37. Adams,T. E., Alpert, S., Hanahan,D. 1987. Non-toleranceand autoantibodies to a transgenicself antigenexpressedin pancreaticfl cells. Nature325:223-28 38. Ornitz, D., Hammer,R., Messing,A., Palmiter,R., Brinster, R. 1987.Science. In press 39. Small, J. A., Khoury, G., Jay, G., Howley,P. M., Scangos, G. A. 1986. Early regions of JC virus and BK virus inducedistinct andtissue-specific tumorsin transgenic mice. Proc. Natl. Acad. Sci. USA83:8288-92

Annual Reviews

Annu. Rev. Immunol. 1988.6:25-48. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

TRANSGENICMICE AND ONCOGENESIS 40. Small, J. A., Scangos,G. A., Cork,L., Jay, G., Khoury, G. 1986. The early region of humanpapovavirus JC inducesdysmyelination in transgenicmice. Cell 46:13-18 41. Barbacid, M. 1986. Humanoncogenes. In ImportantAdvancesin Oncology,ed. V. DeVita, S. Hellman,S. Rosenberg, pp. 3-22. Philadelphia:Lippincott 42. Quaife,C. J., Pinkert, C. A., Ornitz, D. M.,Palmiter,R. D., Brinster, R. L. 1987. Pancreatic neoplasia induced by ras expressionin acinar cells of transgenic mice. Cell 48:1023-34 43. Andres, A. C., Sehonenberger,C. A., Groner, B., Henninghausen, L., LeMeur,M., Gerlinger, P. 1987. Ha-ras oncogeneexpression directed by a milk protein gene promoter: Tissue specificity, hormonalregulation, and tumor inductionin transgenicmice.Proc.Natl. Acad. Sci. USA84:1299-1303 44. Metcalf, D. 1985. The granulocytemacrophage colony stimulating factors. Science 229:16-22 45. Lang, R. A., Metcalf, D., Gough,N. H., Dunn, A. R., Gonda, T. J. 1985. Expressionof a hemopoieticgrowthfactor eDNA in a factor dependentcell line results in autonomous growth and tumorigenicity. Cell 43:531-42 46. Rosenthal,A., Lindquist, P. B., Bringman,T. S., Goeddel,D. V., Derynck,R. 1986.Expressionin rat fibroblasts of a humantransforming growth factor c~ cDNA results in transformation.Cell46: 301-9 47. Hapel, A. J., VandeWonde,G., Campbell, H. D., Young,I. G., Robbins, T. 1986. Generationof an autocrine leukemia using a retroviral expression vector carrying the interleukin-3 gene. Lymphokine Res. 5(4): 249~50 48. Lang, R. A., Metcalf, D., Cutherbertson, R. A., Lyons,I., Kelso, A., Kannourakis,G., Williamson,J., Klintworth, G., Gonda,T. J., Dunn,A. R. 1987. Transgenic mice expressing a haemopoieticgrowth factor gene (GMCSF)develop an accumulationof activated macrophages, blindness and a fatal syndromeof tissue damage.Submitted 49. M/iller, R. 1986.Cellular and viral fos genes: Structure, regulation of expression and biological properties of their encoded products. Biochem. Biophys. Acta 823:207-25 50. Jenuwein,T., M/iller, R. 1987. Structure-function analysis offos protein: A single aminoacid changeactivates the immortalising potential of v-fos. Cell 48: 647-57

47

51. Riither, U., Garber, C., Komitowski, D., Muller, R., Wagner,E. F. 1987. Deregulatedc-fos expressioninterferes with normalbone developmentin transgenie mice. Nature325:412-16 52. Kelly, K., Siebenlist, U. 1985. Therole of c-mycin the proliferation of normal andneoplasticcells. J. Clin. Immunol. 5: 65-77 53. Studzinski,G. P., Brelvi,Z. S., Feldman, S. C., Watt, R. A. 1986. Participation of c-myc protein in DNAsynthesis of humancells. Science 234:467-70 54. Cory, S. 1986. Activation of cellular oncogenesin haemopoietic cells by chromosome translocation. Adv. CancerRes. 47:189-234 55. Stewart,T. A., Pattengale,P. K., Leder, P. 1984. Spontaneousmammary adenocarcinomas in transgenic mice that carry and express MTV/mycfusion genes. Cell 38:627-37 56. Adams,J. R., Harris, A. W., Pinkert, C. A., Corcoran,L. M., Alexander,W.A., Cory, S., Palmiter, R. D., Brinster, R. L. 1985. The c-myc oncogenedriven by immunoglobulin enhancers induces lymphoid malignancy in transgenic mice. Nature 318:533-38 57. Leder,A., Pattengale,K., Kuo,A., Stewart, T. A., Leder, P. 1986.Consequence of widespreadderegulationof the c-myc gene in transgenic mice: Multiple neoplasms and normal development. Cell 45:485-95 58. Corcoran,L. M., Cory, S., Adams,J. A. 1985. Transposition of the immunoglobulin heavychain enhancerto the myc oncogene in a murine plasmacytoma. Cell 40:71-79 59. Grosschedl,R., Weaver,D., Baltimore, D., Costantini, F. 1984. Introductionof a # immunoglobulin gene into the mouse germline: Specific expressionin lymphoid cells andsynthesisof functionalantibody. Cell 38:647-58 60. Tidmarsh,G., Dailey, M., Whitlock,C., Pillemer, E., Weissman,I. L. 1985. Transformed lymphocytes from Abelson-derivedmice express levels of a B lineage transformationassociated antigen elevated from that foundon normal lymphocytes. J. Exp. Med. 162: 142130 61. Whitlock,C. A., Tidmarsh,G. F., MUller-Sieburg, C., Weissman,I. L. 1987. Bonemarrowstromalcell lines with lymphopoieticactivity expresshighlevels of a pre-B neoplasia-associatedmolecule. Cell 48:1009-21 62. Morse,H. C., Tidmarsh,G. F., Holmes, K. L,, Fredrickson, T., Hartley, J., Pierce, J., Langdon,W.Y., Dailey, M.,

Annual Reviews

Annu. Rev. Immunol. 1988.6:25-48. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

48

CORY & ADAMS

Weissman, I. L. 1987. Expressionof the line. Nature322:748-50 6C3 antigen on murine hematopoietic 71. Dotto, G. P., Gilman, M. Z., Maruneoplasms. J. Exp. Med.165:920-25 yama,M., Weinberg,R. A. 1986. c-myc and c-fos expressionin differentiating 63. Braun, J., Forouzanpour,F., King, L., Teheranizadeh,T., Bray, M., Kliewer, mouseprimary kerotinocytes. EMBO J. ÷ S. 1986. B-Lyl cells: ImmortalLy-1 5:2853-57 B lymphocytecell lines spontaneously 72. Bernard, O., Cory, S., Gerondakis,S., arising in maturesplenic cultures. ImmuWebb,E., Adams,J. M. 1983. Sequence nol. Rev. 93:5-21 of the murine and humancellular myc 64. Hayakawa,K., Hardy, R., Herzenberg, oncogenesand two modesof mye transcription resulting from chromosome L. A., Herzenberg, L. A. 1985. Progenitorsfor Ly-1Bcells are distinct from translocation in B lymphoidtumors. progenitors for other B cells. J. Exp. EMBOJ. 2:2375-83 Med. 161:1554~58 73. Nishikura,K., Ar-Rushidi,A., Erikson, 65. Alexander,W., Schrader, J. W., Adams, J., Watt, R., Rovera, G., Croce, C. M. J. M. 1987. Expression of the c-myc 1983.Differential expressionof the normal and of the translocated humanconcogene under control of an immunoglobulin enhancer in E#-myctransmyc oncogenesin B cells. Proe. Natl. genic mice. Mol. Cell. Biol. 7: 1436Acad. Sci. USA80:4822-26 74. Leder,P., Battey,J., Lenoir, G., Moulding, L., Murphey,W., Potter, H., Stew66. Langdon,W. Y., Harris, A. W., Cory, S., Adams,J. A. 1986. Thec-myc oncoart, T., Taub, R. 1983. Translocation gene perturbs B lymphocyte develamong antibody genes in human opmentin E#-myctransgenic mice. Cell cancer. Science 222:765-71 47:11-18 75. Rabbits, T., Forster, A., Hamlyn,P., 67. Harris, A. W.,Pinkert, C. A., Crawford, Baer, X. 1984. Effect of somaticmutaM. C., Brinster, R. L., Adams,J. M. tion within translocated c-mycgenes in 1987. The E#-myctransgenic mouse:A Burkitt’s lymphoma.Nature 309: 592modelsystem for high incidence spon97 taneous lymphoblastic lymphomaand 76. Cory, S., Bernard,O., Bowtell,D., Schleukemiaof early pre-Bcell origin. Subrader, J. W.1987. Murinec-myc retromitted viruses alter the growthrequirementsof 68. Vaux,D. L., Adams,J. M., Alexander, myeloidcell lines. Oncogene1:61-76 W.S., Pike, B. L. 1987. Immunological 77. Rapp,U., Cleveland,J., Brightman,K., competence of B cells subjected to Scott, A., Ihle, J. 1985. Abrogationof IL-3 and IL-2 dependence by recomconstitutive c-rnyc oncogeneexpression in E#-myctransgenic mice. J. Immunol. binant murineretroviruses expressingvIn press myc oncogenes. Nature 317:434-38 68a. Bubbers,J. E. 1984. Identification and 78. Sinn, E., Muller, W., Pattengale, P., linkage analysis of a gene, Rcs-I supTepler, I., Wallace,R., Leder, P. 1987. pressing spontaneous SJL/J lymphoma Coexpression of MMTV Iv-Ha vas and expression.J. Natl. CancerInst. 72: 441MMTV I c-myc genes in transgenic mice: 46 synergistic action of oncogenesin vivo. 69. Coppola, J. A., Cole, M. D. 1986. Cell 49:465-75 Constitutive c-myconcogeneexpression 79. Dickson,C., Peters, G. 1987. Potential oncogeneproduct related to growthfacblocks mouse erythroleukemia cell differentiation but not commitment. tors. Nature326:833 Nature 320:760-63 80. Lee, W.-H., Bookstein, R., Hong, F., Young,L.-J., Shew,J.-Y., Lee, E. Y.-H. 70. Dmitrovsky,E., Kuehl, W.M., Hollis, G.F., Kirsch,I. R., Bender,T. P., Segal, P. 1987. Humanretinoblastoma susS. 1986. Expression of a transfected ceptibility gene: Cloning,identification humanc-myconcogeneinhibits differand sequence. Science 235:1394-99 entiation of a mouseerythroleukemia

Annu. Rev. Immunol. 1988.6:25-48. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Annu. Rev. Immunol. 1988.6:25-48. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Annual Reviews www.annualreviews.org/aronline

Annu. Rev. Immunol. 1988.6:49-83. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

Ann. Rev. lmmunol. 1988. 6 : 4943 Copyright © 1988 by Annual Reviews Inc. All rights reserved

KININ FORMATION: MECHANISMS AND ROLE IN INFLAMMATORY DISORDERS David Proud Division of Clinical Immunology,Johns Hopkins University School of Medicine at The Good Samaritan Hospital, 5601 Loch Raven Boulevard, Baltimore, Maryland 21239

Allen P. Kaplan Division of Allergy, Rheumatologyand Clinical Immunology, State University of NewYork, Stony Brook, NewYork 11794-8161

INTRODUCTION Bradykinin and lysylbradykinin are potent vasoactive peptides liberated from ~2 globulins, called kininogens, by the actions of various proteases, known collectively as kininogenases. The pharmacologic properties of kinins--including their abilities to increase vascular permeability, to cause vasodilatation and pain, to contract most smooth muscle preparations, and to stimulate arachidonic acid metabolism--have led several investigators to suggest that these peptides may be important inflammatory mediators in humans(1-3). Only in recent years, however, with improved assay technologies and an increased awareness of the mechanismsregulating kinin levels, has meaningfuldirect evidence to support a role for the kinin system in humaninflammatory disorders begun to accumulate. In the present chapter we review our current knowledgeregarding the three different pathwaysthat maylead to kinin formation in inflammatory events: (a) Generation of bradykinin as a result of the activation of the Hagemanfactor-dependent pathways and production of plasma kallikrein, (b) the generation of lysylbradykinin by tissue kallikreins, and (c) the potential role of cellular proteases in kinin formation. Wealso critically 49 0732-0582/88/0410-0049502.00

Annual Reviews 50

PROUD & KAPLAN

evaluate the evidence of activation of one or more pathways and the involvcment of kinins during inflammatorydisordcrs in humans.

Annu. Rev. Immunol. 1988.6:49-83. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

MECHANISMS

OF KININ

FORMATION

Hageman Factor Dependent Pathway (4) The formation of bradykinin in humanplasmais dependentuponthe interactionof certain negativelychargedsurfaces withthree plasmaproteins; namely,FactorXII (Hageman factor) (5), prekallikrein(6-8), high molecularweight (HMW) kininogen(9-11) (Figure 1). These proteinsare also the initiating factors requiredfor the intrinsic blood coagulationandfibrinolytic pathways.In plasma,prekallikreinandHMW kininogencirculate as a complex with1 : 1 molarstoichiometry (12), while Hageman factor is not complexed.These proteins bind to initiating surfaces, the Hageman factor becomes activated, prekallikreinis converted to kallikrein, andHMW kininogenis digested to release the vasoactive peptidebradykinin(Figure1). A secondmajorplasmasubstrateof activated Hageman factor, factor XI, also circulates boundto HMW kininogen(13); it is convertedto factorXIa(14), whichcontinuesthe intrinsic coagulationcascade. Hageman factor (HF)(coagulationfactor XII) is a single-chainglobulin of molecularweight80,000(15). It is activateduponcontactwithcertain

surfoce

FACTOR Xl HMWKininogen ~ [

"PRO"-KALLIKREIN Intr~cellulorEnzymes ~ Plosmin PIosmoKollik~’~in

~

TISSUEKALLIKREIN(SECRETED)

FACTOR ~IA

LMWKININOGEN1:~] PREKALLIKREIN - ~ KALLIKRF’IN

i COAGULATION

~<~N~NO~N ~ BRAD¥~:!N, IN IH~w

I

LYSYLBRADYKININ (KALLIDIN)

Corboxypeptidose N (C/aN)

l,~m/nopeptidose

DES -ARC9- BRAOYKININ~ )RRAOYKININ)

l

l ngiotensin Converting Enzyme Other Plosmo Proteoses

INACTIVEPEPTIDES,AMINOACIDS Figure 1

Mechanisms of bradykinin formation

by plasma and tissue

pathways.

Annual Reviews

Annu. Rev. Immunol. 1988.6:49-83. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

KININS AND INFLAMMATION

51

negativelychargedsurfaces, for example,glass, silicates, Lipid Aof endotoxin, dextran sulfate, sulfatides, and heparin. TheHFbecomescleaved (16, 17) and expresses enzymaticactivity against low molecularweight synthetic substrates as well as against a numberof plasmaproteins. As seen in Figure1, the major protease capable of activating Hageman factor i~ kallikrein (18). Otherweakeractivators are plasminor factor XIa, each of whichis formedas a consequenceof Hagemanfactor activation. If one examinespurified preparations of Hageman factor and prekallikrein, minimalenzymaticactivity is seen if precautions are taken to eliminate contaminating activating enzyme. Whenmixed and exposed to macromolecular anionic materials--"surface"--rapid activation of both proteins results. However,surface-dependentactivation of Hageman factor can occur in the absence of prekallikrein, and the activated Hageman factor can convertfactor XI to factor XIa so that coagulationcan proceed. Congenitalprekallikrein deficiency(Fletcher Trait) behavesin this fashion (19, 20). Bycontrast HF-deficientplasmadoes not activate uponcontact with a surface. Thus, Hageman factor is essential for contact activation, while prekallikrein contributes a prominentaccelerating action which, however,is not absolutely required. Hageman factor can autoactivate (21-23), and this mayexplain the gradual evolution of coagulantactivity in prekallikrein deficient plasma. The kinetics of appearanceof activity in purified Hageman factor when exposedto a surface ha~e been analyzed by an iterative computermodel (24), an explicit secondorder kinetic model(25), and by demonstration exponentialinitial rates (22), all consistent with an autoactivationmechanism. Activated Hageman factor (HFa)has been shownto cleave native Hageman factor to generate more HFain the cases of both rabbit (23) and humanproteins (22, 26). The dilemmaposed by an autoactivation mechanism is whereor howthe initial activated moleculesare created so that native HFmaybe digested. Someproposals consideredthe possibility that surface binding of HFcould create an active site by a conformational changeprior to any cleavage (27, 28). Other workers suggested that substrate-induced conformational change occurs in HFwhenHFplus substrate is boundto the surface (29, 30). However, proof of the creation of active sites prior to proteolysisis very difficult to obtain. Therates of activation seen in mixturesof HFand prekallikrein accelerate rapidly due to the positive feedback.Tankersley& Finlayson(24) estimatedthe kinetic constants for the componentreactions and calculated that, at plasma concentrations of HFand prekallikrein, the introduction of a surface in whichonemoleculeof active enzymeis present per milliliter (5 x 10-13 total protein) will yield 50%activation in 13 sec. Since the best preparations of HFhave about 0.02%active enzyme,it is not possible to

Annual Reviews

Annu. Rev. Immunol. 1988.6:49-83. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

52

PROUD & KAPLAN

achieve or measuresuch infinitesimal activation. Wehave used a rapidly acting chloromethylketone to destroy all traces of activated HFin preparations of HFand reducedenzymaticactivity to less than 1/4200of that of HFa(31). Eventhen, HFcould autoactivate and convert prekallikrein to kallikrein, albeit with a very low initial rate. Wethen proposedthat enzymaticactivity containedin native HF(if any) is too low to measure and that contact activation mayinvolve participation of extremelylow levels of active enzymethat are continuouslypresent. Thepossibility that native HFhas enzymaticactivity analogousto that seen in trypsinogen(32) (comparedto trypsin) is not excludedbut is subject to the aforementioned constraints. Thesurface appears to accelerate contact activation in two ways. It causesa conformationalchangein native HFsuch that its rate of cleavage is increased(33). This suggeststhat boundHFis a better substrate regardless of whetherHFa(autoactivation) or kallikrein effects its cleavage. addition, the catalytic effect of the surface maybe to create a local milieu in whichreversible binding of protein componentsresults in an increase in local concentration,therebyacceleratingtheir rate of interaction. Once someactivated HFconverts prekallikrein to kallikrein, the kallikrein rapidly activates the bulk of HF.Since the latter is far morerapid, this pathwayis kinetically dominant;hence the thickened arrow in Figure 1. Recentevidenceindicates that kallikrein also has to bind to the surface to activate HFwith maximalefficiency (34, 35). The idea of a reciprocal reaction betweenthe zymogenand activated forms of Hageman factor and prekallikrein was complicatedby the realization that yet another component is required for optimal contact activation (36, 37). The plasmaof patients not deficient in either Hageman factor or prekallikrein had a markedlyprolongedkaolin-inducedpartial thromboplastintime and generated no bradykinin. This led to the identification of HMW kininogen as a non-enzymaticcofactor that augments the activation of both prekallikrein and factor XI by activated Hageman factor and increases the rate of Hageman factor activation by kallikrein (38, 39). One critical observation regarding the mechanismby which HMW kininogen functions as a cofactor is the demonstrationthat both prekallikrein (12) and factor XI (13) circulate in plasma bound to kininogenand that these complexesare boundto initiating surfaces via the HMW kininogen moiety. In plasma, HMW kininogen augments the attachmentof prekallikrein and factor XI to surfaces such as kaolin (40, 41). Whenthe HMW kininogenis cleaved, this effect is augmented(42). Thus, HMW kininogen maybe an activatable cofactor (42) although the magnitudeof this effect is uncertain. Adhesiveglycoproteinssuch as

Annual Reviews

Annu. Rev. Immunol. 1988.6:49-83. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

KtNINS

AND INFLAMMATION

53

fibrinogen may compete with HMW kininogen for binding to initiating surfaces, e.g. antigenic fibrinogen in clot promoting surfaces becomes undetectable after l0 rain when HMW kininogen is present, an effect not observed in HMW kininogen deficient plasma. Perhaps cleaved HMW kininogen is a more effective competitor. Yet in buffer systems (41), prekallikrein and factor XI bind to kaolin equally well in the presence or absence of HMW kininogen, but activation of each by HFa is still accelerated upon addition of HMW kininogen. Thus, aside from quantitative binding effects seen in plasma, HMW kininogen must also place the substrates of activated Hagemanfactor upon the surface in a conformation that facilitates their cleavage. Moredifficult to explain is the ability of HMW kininogen to augment the cleavage and activation of Hageman factor by kallikrein since it has no demonstrableeffect uponthe enzymatic activity of kallikrein (using synthetic substrates) and has no knowninteraction with Hagemanfactor. The dissociation constant for the prekallikrein (kallikrein)-HMW kininogen complex is 15 nM(43), and plasma concentration, about 10-20%of prekallikrein circulates free (44). Thus, kallikrein formed by activation of surface-bound prekallikreinHMW kininogen-complexescan dissociate from the surface. It is then able to digest Hagemanfactor molecules on the same or other particles (41, 45). In this fashion, contact activation is disseminated along the surface. Thus, the effective ratio of kallikrein/Hagemanfactor is increased in the presence of HMW kininogen, and this may account for the augmentation seen. The magnitude of this contribution of HMW kininogen varies depending upon the nature of the surface used and whether the reaction is studied using plasma or purified components. Hageman factor (Figure 2) is initially cleaved within a disulfide bridge so that a single chain of 80,000 is converted to a two-chain enzymein which a heavy chain of 50,000 is disulfide linked to a light chain of 28,000 (46). Noloss in size results, and the enzymeis called HFa(or ~ HFa). The binding site for the surface is on the heavy chain (47), and the light chain contains the active enzymatic site. Further cleavage by kallikrein digests HFaat two sites (2 and 3) in sequence (48) to form HFf (or fl HFa) in which a heavy chain 28,000 is disulfide-linked to a light chain of either 2500 or 500 (64). mixture of these two forms accounts for the doublet typically seen in alkaline disc gels (12) or unreduced SDSgels (16). Enzymesother than kallikrein are able to cleave and activate Hageman factor, including plasmin (17, 33, 49) and factor XIa (18), but efficiencies of these reactions are muchlower. The cleavage of Hageman factor by HFaduring autoactivation is potentially of greater significance CLEAVAGE OF PROTEINS DURING CONTACT ACTIVATION

Annual Reviews 54

PROUD & KAPLAN SURFACE BINDINGSITE HzN ...........................

~

SITE 4 (?) mmHFo---~40,OOOMW i.-~I ~ activefragment ~ I L

............

-I I

Annu. Rev. Immunol. 1988.6:49-83. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

SITE, (~53.__.__~)

,.,N..~Ar V~OI g-

~l / SITE.~2 (334__.~)

SITE~_.(343)

V ~ Thr Mt r r - e-Se-Se-Leu-Ser-Lys-Arg-Leu-Arg-Gly-Cys-Ser-Leu-Pro-Gly-Asn

Gly"GIn

HOOCSer-VoI-Thr

Cys

(~

Fi#urc 2 Cleavagesites of Hagcman factor during activation. Initially, cleavagesitc converts the single chain HF to a two-chain disulfide linked enzyme (HFa). Theheavychain contains the surface binding site andthe light chain has the active site. TheScr, Asp, His forming the "charge relay" of scrine proteasesa~e circled. Cleavageat sites 2 and then converts HFato HFr. The light chain of HF~(1) is 18 amino acids (site 1 to site 2) and

light chainof HFr(2) is 8 amino acids(site 3 to site than these reactions. Autoactivation produces, in addition to HFa and HFr, a third form in which the serine protease domainis linked to a 12,000 mol wt fragment of heavy chain to give a molecule ofmol wt 40,000 before reduction (17, 26; Figure 2), but kallikrein rapidly converts it to HFr. The complete amino acid sequences of the heavy chain of HFa (50) and of both chains of HFr (51) is nowknown.The latter study found the light chain of HFf to have nine amino acids presumably corresponding to the short segment remaining after cleavage at site 3. The heavy chain was found to possess a variety of structural domains with homologyto other proteins. Twoshort domains resemble fibronectin, two regions are homologous with epidermal growth factor, and a kringle structure is seen, as reported in plasminogen, tissue plasminogen activator, urokinase, and prothrombin. The light chain of HFa(or heavy chain of HFr) containing the active site has a high degree of homology with tissue plasminogen activator and, in decreasing order, with urokinase, plasmin, trypsin, factor X, and factor IX. Strong homologywith proteins involved in fibrinolysis is apparent. More recently, the complete amino acid sequence has been deduced using factor XII cDNAisolated from a cDNAlibrary prepared from human liver mRNA(52). HFr lacks the binding site for the surface and, once formed, is released

Annual Reviews

Annu. Rev. Immunol. 1988.6:49-83. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

KININS AND INFLAMMATION

55

into the fluid phase. It is not a coagulationfactor (it possessesonly 2-5% of the coagulantactivity of HFa)(16, 17), becausefactor XI activation strictly surface dependent(53). However, HFfis an effective prekallikrein activator (16, 17,. 53) and can continue to lead to bradykinin formation until Cl-inactivator (CI-INH)binds andinactivates it (54, 55). Prekallikrein is a single chain a globulin with two forms at mol wts 85,000and88,000(56), both of whichare present in all plasmastested. manyas seven formswith isoelectric points 8.5 and 8.9 can be identified by isoelectric focusing(57). Prekallikrein is convertedto kallikrein cleavage within a disulfide bridge such that a heavychain of 52,000 is disulfide linked to light chains of either 33,000and 36,000(one for each molecularform) (56, 58). Theheavychain interacts with the surface binds to HMW kininogen (34), while the light chain has the enzymatic active site (56). Purified light chainretains enzymatic activity on synthetic substrates (34) or in fluid-phase kinin generationbut is ineffective as surface-dependentcoagulationfactor. Factor XI is unusual amongcoagulation factors in that it consists of two identical disulfide-linked chains (59, 60). Uponactivation each chain is cleavedto yield disulfide-linkedheavyandlight chains; thus, the active enzymehas four chains. The heavychains bind to the surface (61), while each light chain has an active site (62) and, wheninactivated, can bind one moleinhibitor (60). Also, it has recently beenshownthat the molarratio of binding of HMW-kininogen to factor XI is 2 : 1 (63). The HMW kininogenis cleaved by plasmakallikrein at two sites (LysArg, and Arg-Ser)to liberate the nonapeptidebradykinin. Bothcleavages occur within a disulfide bridge (64~6) so that kinin-free HMW kininogen consists of a heavychain of molwt 42,000-65,000disulfide linked to a light chain variously reported as havinga molwt of 56,000-62,000.Further proteolysis of the light chainby kallikrein convertsit to a light chainof 45,000-47,000(64, 66, 67). Theheavy chain of HMW kininogen, which identical to the heavy chain of low molecular weight (LMW) kininogen (64, 68, 69), has beenshownto inhibit cysteine proteases (70-72)such cathepsin L, papain, or platelet calpain 2 (73). In fact, cDNA analysis a2 thiol proteinase inhibitor demonstratedthat it is identical to LMW kininogen(71). Thelight chain of H MW kininogenis the coagulantportion of the molecule(64, 74) andpossessesa binding site for the surface (74) as well as a bindingsite for the heavychain of prekallikrein (12, 43, 44, 74, 75) or factor XI (12, 74, 76) (Figure 3). Thesebinding constants beenestimated to be 3.4 × l07 M-1 and 4.2 x 108 M-1, respectively (74). CONTROL MECHANISMS Regulation of the Hagemanfactor pathways occurs by both intrinsic and extrinsic mechanisms.Cleavageof HFato

Annual Reviews 56

PROUD

& KAPLAN BRN}YKININ ~

CYSTEINE

PROTEASE

2

INHIBITOR H2N

I 5

Annu. Rev. Immunol. 1988.6:49-83. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

CLEAVAGE

t

SURFACE 3 SITE

LluMetLys Ar(]---ArgSer

BINDINGSITE FOR

BINDING PREKALLIKREIN OR FACTOR Xl

I S

RESULT

SITE I

¯ ¯ ¯

FORMS"NICKED" HMWKININOGEN AUGMENTATIONOF COAGULATIONCOFACTORFUNCTION REDUCTION~BRADYKININ AT C-TERMINUS OF H CHAIN

SITE 2

¯ ¯

RELEASEOF 8RADYKININ REDUCTION~62,000 H CHAIN ÷ 56,000 L CHAIN

SITE 3

¯

CONVERTS L CHAIN FROM56,000 TO 47,000 AND RELEASES PEPTIDEOF 8,000 RETAINS COAGULANT ACTIVITY THE LOCATION OF THIS SITE IS UNCERTAIN,BUTIS DEPICTED AT AMINO TERMINALEND OF L CHAIN

¯ ¯

Figure 3 Mechanism of digestion

of HMW-kininogcn to release

bradykinin.

functional domains of the molecule are shown and the consequences of cleavage is described.

The various at each site

HFf is one example of an intrinsic control in which coagulation via the surface dependent activation of factor XI is limited while bradykinin generation via fluid phase activation of prekallikrein can continue. Likewise, the digestion of kinin-free HMW kininogen light chain by factor XIa limits its coagulant activity (77). Extrinsic controls are provided by plasma inhibitors of each enzyme. The only major plasma inhibitor of HFa or HFr is CI-INH(54, 55, 78, 79). There are two main kallikrein inhibitors in plasma; namely a2 macroglobulin (80) and CI-INH (81, 82). kallikrein is added to plasma, approximatelyhalf is boundto each inhibitor at equilibrium (83, 84). However,when a surface such as kaolin is added to plasma, 70-80%of the kallikrein formed is bound to CI-INH(85); the mechanismresponsible for this change in inhibition ratio is unknown. Factor XIa is inhibited primarily by a~ antitrypsin (86-88) and CI-INH (78), while plasmin is inhibited by a2 antiplasmin (89, 91) and a2 macroglobulin (92-94). AntithrombinIII (ATIII) accounts for only a few percent of plasma inhibition of each of these factors, but its activity is augmented in the presence of heparin. Although this augmentation is less than that shownwhen the kinetics of thrombin inactivation are examined, there is clearly a prominent effect, the magnitudeof which is controversial. These reactions need to be reassessed using purified components,and the rate of inhibition of each enzymeshould be carefully determined in heparinized

Annual Reviews

Annu. Rev. Immunol. 1988.6:49-83. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

KININS

AND INFLAMMATION

57

vs citrated plasma.For such studies the dose of heparin and definition of the activity of the fraction usedwill be critical. Bradykininis an exceedinglypotent vasoactive peptide whichcan cause venular dilatation, increased vascular permeability, hypotension, constriction of uterine and gastrointestinal smoothmuscle, constriction of coronaryand pulmonaryvasculature, bronchoconstriction,and activation of phospholipaseA2to augmentarachidonic acid mobilization. Thus, its regulation is of prime importance, and plasma contains a variety of enzymesthat serve this function. Theaminoacid sequenceof bradykinin and the mechanisms by whichit is degradedare shownin Figure 4. First, the C-terminalarginine is removedby carboxypeptidase N(95, 96) to leave the residual octapeptide. Thedes-Arg9 bradykininthus formedis inactive in the skin or gastrointestinaltract but displaysactivity in severalin vitro vascular preparations (3). Separate receptors for these two moieties have 9 bradykinin)and B2(bradykinin) beenproposedand are called B1(des-Arg (97). Therate of Arg removalin serum, however,far exceedsthe rate plasma(98), whilethe level of carboxypeptidase N is unaltered. It appears that either a cofactor that augmentsthe activity of carboxypeptidase Nor a carboxypeptidaseB-like enzymeis generated during coagulation. DesArg9 bradykinin is then degraded, by angiotensin-convertingenzyme,to the pentapeptide Arg-Pro-Pro-Gly-Pheplus the tripeptide Ser-Pro-Phe (99). The C-terminal phenylalanine is then cleaved from each peptide, leaving Arg-Pro-Pro-Gly and Ser-Pro. The Ser-Pro is then digested to Ser and Pro while the C-terminal Gly is removedfrom the tetrapeptide. The final plasmadegradationproductsof bradykinin are, therefore, one mole each of Arg-Pro-Pro,Gly, Ser, Pro, and Arg, plus two molesof phenylalanine.

Arg Pro Pro Gly Phe Ser Pro Phe Arg

Brodykinin

CPN or CPB

~ Arg Pro Pro Gly Phe Set Pro Phe + ArcJ des - Arg 9- 1,4CE

Brodykinin

Arg Pro Pro Gly Phe + Set Pro Phe 7 enzymes (s Arg Pro Pro + Gly + Phe

? enzyme(s) Ser + Pro + Phe

Figure 4 Amino acid sequence of bradykinin and products obtained upon digestion human serum.

in

Annual Reviews PROUD & KAPLAN

Annu. Rev. Immunol. 1988.6:49-83. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

ASSESSMENT OF CONTACT ACTIVATION IN HUMAN DISEASE The

Hageman

factor-dependent pathways described herein are ~aot likely to contribute to thrombosis and hemostasis in a major way. Thus, although activation of Hagemanfactor leads to in vitro coagulation, in most thrombotic disorders activation of the extrinsic pathway occurs either alone or in addition to Hagemanfactor activation, and the effects of tissue factor predominate. Certainly no thrombosis is associated with hereditary angloedema, a disorder in which contact activation is prominent. Likewise, patients deficient in Hagemanfactor, prekallikrein, and HWM kininogen have no obvious bleeding diathesis, and factor XI deficient patients have a bleeding disorder that is highly variable and often mild. Observations such as these have led to considerations of physiologic mechanisms of factor XI activation that bypass the other contact activation components, but this remains unsettled. Most evidence suggests that the importance of the cascade relates to the pathogenesis of inflammatoryreactions, the local control of blood flow (in which bradykinin functions as a hormone), and perhaps control of blood pressure. To assess the Hagemanfactordependent pathways in humandisease, we would like ideally to measure each active enzyme, demonstrate cleaved HMW kininogen, and assay bradykinin. These determinations are difficult because the enzymesand the bradykinin are rapidly inactivated and often one has to determine the level of residual proenzyme. Thus, one can quantitate Hagemanfactor, prekallikrein, or HMW kininogen in plasma by coagulant assay, (100), immunologic determination of antigen levels (101-103), or by in vitro activation and evolution of enzymatic activity (104-107). Shifts in electrophoretic mobility indicative of complex formation between enzymes and their inhibitors can be assessed qualitatively. Thus far, monoclonal a~tibodies reactive solely with the active site of enzymeshave not been developed, although DeAgostini et al (108) have described a monoclonal antibody reacting with a neoantigen in the kallikrein-C1 INH complex. The aboveassays are useful if significant activation has occurred, leading to substrate depletion. But if only a small percentage conversion of each factor takes place, it is likely to be missed. Assays have recently been developed by Harpel for enzyme-inhibitor complexes based on the assay for plasmin-~2 antiplasmin complexes (109). These include double antibody ELISA methods for activated Hagemanfactor-C1 INH (110), kallikrein-C1 INH(111), and kallikrein--~2 macroglobulin complexes (85), which can detect as little as 1%activation of any of the components. Bradykinin can be determined by radioimmunoassay (112), and a radioimmunoassayfor des-Argg-bradykinin has been developed (113).

Annual Reviews

KININS ANDINFLAMMATION 59

Annu. Rev. Immunol. 1988.6:49-83. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

Tissue Kallikreins Tissue kallikreins are single chain, acidic glycoproteins physicochemieally and immunologically distinct from plasma kallikrein. Thus, they have a reported tool wt range of 25,000 to 43,000 and isoelectric points close to pH4.0 (114, 1 ! 5). WhileHMW kininogenis clearly the preferred substrate for plasma kallikrein, tissue kallikreins readily release kinin from both HMW kininogen and LMW kininogen (116, 117). Immunoreactive tissue kallikrein has been detected in both rodent (118-120) and human(121), plasma, but most, if not all, of this material apparently represents biologically inactive kallikrein destined for clearance by the kidneys (119). Whilesomesystemic role for tissue kallikreins cannot be ruled out, therefore, it seems likely that these enzymesfunction principally in the local environmentof their tissues of origin. Originally tissue kallikreins were thought to derive only from the major exocrine organs such as the pancreas, salivary glands, and kidneys. More recently, however, kallikreins were discovered to be more widely distributed in tissues such as the gut (122) and the prostate gland (123). Althougha report that tissue kallikrein was also derived from the endocrine portion of the pancreas (124) has been vigorously disputed (125127), morerecent workdescribing the presence of kallikrein in the pituitary gland (128-~130)leaves little doubt that these proteases are not restricted to exocrine tissue. , Within a given species, the tissue kallikreins from all organs are immunologically identical (131-133). Furthermore, recent eDNAcloning and sequence analysis studies have shownthat humanpancreatic and renal kallikreins have identical aminoacid sequences, with the active enzymein each case consisting of 238 residues (134, 135). In addition, the same mRNA is expressed in the pancreas, kidney, and sublingual gland (134). Although in both mouseand rat, kallikrein activity is associated with the highly conserved members of a large multigene family located on chromosome 7 (136--138), studies suggest that there are no more than two or three closely related genes in humans(135). Kallikreins are synthesized in all tissues examinedto date in the form of the preproenzyme. Whenstudied by conventional biochemical techniques, however,kallikrein is detected in someorgans, such as the pancreas (139, 140) and kidney (141) primarily in the form of prokallikrein, while in submandibular gland only the active kallikrein is found (142). Further posttranslational differences also occur with regard to glycosylation of tissue kallikreins. Isoelectric focussingof purified kallikreins fromdifferent organs usually reveals microheterogeneityin terms of multiple isoelectric

Annual Reviews

Annu. Rev. Immunol. 1988.6:49-83. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

60

PROUD & KAPLAN

forms (115, 143, 144). Treatment with neuraminidase to removesialic acid abolishes this microheterogeneity (143). The distribution of kallikrein in a range of tissues of differing function has led to the suggestion that tissue kallikreins mayplay differing roles depending upon their tissue of origin (145). This is supported by evidence that synthesis and secretion of these enzymesare controlled by different factors in different tissues. It is well established, for example,that restriction of dietary sodiumcauses elevations in urinary kallikrein derived from the kidney (146, 147). This effect maybe mediated by mineralocorticoids, because patients with primary aldosteronism also have elevated urinary kallikrein levels (148), and administration of the synthetic mineralocorticoid fludrocortisone reproduces the effects of dietary sodium restriction (146, 147). Morerecently, parotid salivary kallikrein has been shownto respond similarly to mineralocorticoids and sodium restriction (149). Aldosteronereceptors are present in both the kidney and the parotid gland ’(150), and while it is possible under certain circumstances to dissociate aldosterone and kallikrein levels (151), mineralocorticoids may directly regulate renal kallikrein (152). In other tissues, kallikrein levels appear to be controlled by androgen concentrations. In the rat submandibulargland the tissue content of kallikrein is elevated by testosterone or thyroxine, but no such effects are observed in the kidney (153). The increased kallikrein content of the submandibular gland in response to androgenis due to increased synthesis (154). By contrast, kallikrein levels in the anterior pituitary are considerably higher in female rats than in males (155), and gene expression in this tissue, as determined by mRNA levels, is estrogen, not androgen, dependent (130). The secretion of kallikrein from different tissues is likewise controlled by a spectrumof stimuli. As mentioned above, mineralocorticoids appear to play a role in renal kallikrein secretion, apparently favoring the secretion of the active enzyme (156), while salivary kallikrein secretion is largely under sympathetic control (157, 158). By contrast, pancreatic kallikrein secretion appears to controlled by the vagus nerve and by cholecystokinin (159), but secretion from intestinal goblet cells is largely under cholinergic control (160). This confusing array of control mechanisms and the widespread distribution of kallikreins have led several investigators to suggest that these enzymes cannot serve primarily as kinin-forrning enzymes in all these tissues but must, in somecases, act principally on substrates other than kininogen. For example, it has been suggested that in the pituitary gland kallikrein mayfunction in prohormoneprocessing. The last few years have seen a flurry of effort to search for alternate substrates for tissue kallikreins. It has been suggested that tissue kallikreins can activate prorenin to renin (161,162), but this reaction appears to require noncatalytic quantities

Annual Reviews

Annu. Rev. Immunol. 1988.6:49-83. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

KIN1NS

AND INFLAMMATION

61

kallikrein. Likewise exogenoustissue kallikrein can process atriopeptinogen,the precursorof atrial natriuretic factor (163), but it has also beensuggestedthat kallikrein can inactivate atrial natriuretic peptides (164, 165). Anotherpotential substrate for kallikrein is apolipoprotein 100 whichcan be degradedby urinary kallikrein (166), but the physiological significanceof this reaction remainsunclear. Analternative hypothesis for the functionsof tissue kallikreins in different organsis that they mayserve to producekinins but that the primaryresponse to kinins may vary fromtissue to tissue. In the kidney, for example,kallikreins are synthesized in the connectingtubule (167, 168), and the primarysite kinin bindingis in the cortical andouter medullarycollecting tubule (169). Thus,kinins mayfunction in regulating sodiumexcretion and in regulating the hydro-osmoticresponse to vasopressin (170, 171). In the salivary glands kinins mayalso play a role in regulating the ionic compositionof secretions, or alternatively they mayfunction to control local blood flow (172). Bycontrast, in the intestine kinins mayregulate chloride transport (173, 174) or, perhaps,regulate nutrient absorption(175). Thetissue-specific roles of these enzymesclearly remainsa subject of considerable speculation. Regardinginflammatoryreactions, however,the widespreaddistributions of tissue kallikreins enhancestheir potential for involvementin kinin generation at multiple sites. Likewise, while kallikreins may,indeed, have roles outside of kinin-formation, there can be little doubtof the effectiveness of these proteases as kininogenases. Approximately70%of the total kininogen in humansis LMW kininogen. Unlike plasmakallikrein, therefore, whichfunctions principally on HMW kininogens,the ability of tissue kallikreins to react equally well with HMW and LMW kininogens (116, 117) provides these enzymeswith a larger pool of availablesubstrate. Theinteractionof tissue kallikreins with single-chain kininogens has always been fascinating from the biochemicalstandpoint because these enzymeshydrolyze two dissimilar bonds in the kininogen moleculeto liberate lysylbradykinin(Figure 3), or in a recently described variation, ala4-1ysylbradykinin(176). (This variant, however,apparently displays the samephysiological properties as does lysylbradykinin.) The ability of tissue kallikrein to hydrolyzetwodissimilar bondsandrelease lysylbradykininis still not fully understood.Onesuggestionis that tissue kallikreins mayhavetwoactive sites (177), but little firm evidencesupports this conceptas yet. Tissue kallikreins are knownto contain the normal charge-relay system found in serine proteases, and it has been suggested that the initial hydrolysismayoccur at the Arg-Serbondin kininogen,an idea in keepingwith the anticipated primarysubstrate specificity of tissue kallikreins (115). Althoughtissue kallikreins will hydrolyzemethionyl bonds in small peptides, the preceedingaminoacid sequencemayplay an

Annual Reviews

Annu. Rev. Immunol. 1988.6:49-83. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

62

PROUD & KAPLAN

importantrole. Thebulky leucine residue at position P2 in the kininogenderived peptide Ser-Leu-Met-Lys-bradykinin, for example,is thought to be responsiblefor directing the attack of kallikrein to the methionylbond to release lysylbradykinin (178). Toachievea high catalytic rate of hydrolysis of such a methionylbond, however,the intact kininogenmoleculeis necessary and presumably provides an essential stereochemical conformation. Afinal considerationin understandingthe generationof kinin by tissue kallikreins is the set of factors that controlthe activity of these enzymes. As mentionedabove, in manytissues kallikreins are present mainly in the formof prokallikrein. Whileit is knownthat prokallikrein can be activated by trypsin, plasmin, or even plasmakallikrein, the identity of such an endogenouskallikrein-activating enzymein tissues such as the kidney remains unknown.Onceactivated, however,tissue kallikreins are less susceptible to inhibition by plasmaprotease inhibitors than is plasma kallikrein. Indeed,in humans,only g ~-antiproteaseis a significant inhibitor of tissue kallikrein, and even then inhibition is weakand slow (179). Althoughbovine pancreatic trypsin inhibitor is an effective kallikrein inhibitor in ruminants,an equivalentinhibitor has yet to be isolated from humans.Monovalentcations havebeen reported to inhibit the esterolytic activity of tissue kallikrein (180, 181), but while onereport suggeststhat such ions also inhibit kininogenaseactivity (180), another suggests that they enhancethe kinin-generatingactivity of these enzymes(181). In summary,tissue kallikreins are extremelypotent kinin-generating enzymes,widelydistributed in humans.Their ability to interact with both HMW and LMW kininogens to generate lysylbradykinin and their relative resistance to inhibition enhancestheir potential for involvementin kinin formation during inflammatorydisorders. Cellular Proteases Acommon feature of several types of cells involvedin inflammatoryevents is their high content of proteases. It is clear that, should any of these cellular proteasesdisplay kininogenaseactivity, the potential wouldexist for a direct meansof kinin formation during inflammatorydisorders, independentof plasmaor tissue kallikreins. This concepthas attracted the attention of several investigators whohavestudied the ability of a number of cell types to liberate kininogenase activity. In the case of immediatehypersensitivityreactions, efforts havefocused on the basophil and the mastcell as sources of kinin-generatingenzymes. Preparations of humanleukocytes containing basophils have been shown,

Annual Reviews

Annu. Rev. Immunol. 1988.6:49-83. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

KININS AND INFLAMMATION

63

uponIgE-mediatedstimulation, to release arginine esterolytic activity (182). This activity, measuredby its ability to hydrolyzethe synthetic subs~rate N-~-p-tos3d-L-arginine methyl ester (TAME),showedrelease characteristics similar to those of histamine, suggestingthat the esterase is derived fromthe basophilgranule. Biochemical characterization of this activity showedthat TAME-esterase activity eluted on gel filtration with an apparent mol wt of 1.2 × 10 6 and was associated with both kiningenerating and Hageman factor-cleaving activities 083). The unusually high molecularweightof this entity together with its ability to display several functional activities raises the question of whethermorethan one enzymewasactually beingstudied. In the mastcell high salt concentrations are necessaryto disrupt the interactions of cellular proteases with the proteoglycancomplex(184, 185). Thus, since low salt concentrationswere used in the abovestudies, the apparentmolecularweightmaybe explained by a complexof several enzymesattached to a proteoglycan. In recent preliminarystudies using purified (5%to 84%)basophils, the release TAME-esterase activity in response to an IgE-mediatedstimulus has been confirmed,but not the presence of kinin-generating ability (D. Proud, unpublishedobservations). Antigen challenge of passively sensitized humanlung fragments has beenassociatedwith the generationof three enzymaticactivities: a kininogenase(186), a prekallikrein activator (187), and an enzymethat cleaves but does not activate Hageman factor (188). Generationof these activities in responseto antigen challengefocussedattention on the mast cell as a possible sourceof these enzymes.Prekallikrein activator activity has been detected fromstimulatedmastcells (189) althoughrelatively large numbers of mast cells are neededto generate significant amountsof activity. A Hageman factor-cleaving enzyme,apparently identical to neutrophil elastase, is also released by an IgE-mediatedmechanism from purified mast cells (190). Kininogenase activity is also releasedfromhighlypurified mast cells (191). Althoughit had beenreported that tryptase, the majorneutral protease from humanlung mast cells, does not possess kinin-generating activity (192, 193), recent characterizationhas demonstratedthat the mast cell kininogenaseis, indeed, tryptase. However,this enzymegenerates bradykininoptimallyat pH5.5 (194). Whiletryptase is a relatively weak kininogenase,it is present in mastcells in large amounts.It remainsto be determined whythis neutral protease generates kinin optimally at pH 5.5, but this acidic pHoptimumclearly raises questions regarding the physiologicalrole of tryptase as a kininogenase.Thepropertiesof tryptase suggest that it is not the enzymedescribed fromchoppedlung (186) and raise the possibility that this latter enzymeis not mastcell-derived. Since

Annual Reviews

Annu. Rev. Immunol. 1988.6:49-83. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

64

PROUD& KAPLAN

tissue kallikrein has been detected in bronchoalveolar lavage fluids from asthmatics (195), it is possible that the enzymemeasuredin choppedlung may represent tissue kallikrein released by an indirect mechanismor, perhaps, someother protease yet to be characterized. Calpains are calcium-activated cytosolic cysteine proteases present in manytissues and cells, including reticulocytes, keratinocytes, and platelets (196). Since platelets, for example, can be activated during certain inflammatory states such as asthma (197), a recent report that calpains can generate kinins (198) bears scrutiny. Kininogens are now known to thiol protease inhibitors (72, 199) and have been shownto inhibit platelet calpain (73). This potent inhibitory capacity presumably explains why kinin formation occurs only in a limited and high range of calpain to kininogen molar ratios (0.5 : 1 to 8 : 1). The report that lysylbradykinin liberated from kininog~ns by these enzymessuggests that calpains are the first enzymesother than tissue kallikreins to be able to hydrolyze the dissimilar bonds in kininogens to release lysylbradykinin. Only 20%of the kinin content of the kininogensused in this study was liberated by calpains, however, and it remains to be determined if this is due to subsequent inhibition by kininogens or to the presence of "nicked" two-chain kininogen in these preparations. Thus, more work is required to ascertain that calpains can liberate kinins from single-chain kininogen and to determine if the high molar ratios of calpain to kininogens used by Higashiyamaet al (88) are achieved during inflammatory events in vivo. The first report that neutrophils maycontain a neutral protease capable of generating kinin was in 1967 by Melmon& Cline (200) who incubated intact neutrophils with whole plasma or a crude kininogen preparation. These authors suggested that neutrophils contained both kinin-generating and kinin-destroying activities. While other workers were unable to reproduce these results with intact neutrophils, Movatet al were able to use fragmerited neutrophils with purified kininogens to demonstrate the presence of such a neutral protease from these cells (201). The enzymeappears to be distinct from elastase and cathepsin G (202, 203), but the nature the kinin generated by the purified protease remains uncertain. At this point the relative activities of the kinin-generating and kinin-destroying enzyme during inflammatory events also remain to be determined. In addition to this neutral kininogenase, neutrophils are amongthe cells containing an enzyme capable of generating leukokinins. This enzyme, which is almost certainly cathepsin D, interacts with a precursor, whichis distinct from HMW and LMW kininogens, to liberate pharmacologically active peptides of 21-25 aminoacids called leukokinins (204). This process occurs optimally at acidic pH and is rather slow. Thus, even though all the components of the leukokinin system are present, for example, in

Annual Reviews KININS AND INFLAMMATION

65

ascitic fluid, moreworkis neededto elucidate the role of leukokininsin humaninflammatoryprocesses.

Annu. Rev. Immunol. 1988.6:49-83. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

ROLE

IN

INFLAMMATORY

DISORDERS

Rhinitis Clinical symptoms of thinitis can be inducedby a variety of allergic and nonallergic stimuli. In recent years data havebeenobtainedwhichsuggest that kinins maybe involvedin the pathogeneses of several types of rhinitis. A model of nasal challenge with allergen in which inflammatory mediatorscan be measuredin secretions obtainedby lavage (205) has been used to demonstrate that kinin formation occurs during the immediate response to allergen challenge (206). Kinin generation correlates with the onset of symptomsand with the production of other inflammatory mediatorsincluding histamine, prostaglandin DE(205, 206), and the sulfidopeptide leukotrienes (207). Unlikeplasma, in whichthe measurement of kinins is technically complex(208,209),it is possible to measurekinins simply and reproducibly in nasal lavages collected in the presence of EDTA.In part this maybe due to the large dilution factor created by lavage. Becauseof these advantages,the nasal systemhas also lent itself to biochemicalstudies of the mechanisms of kinin formationand metabolism during the immediateallergic response. The demonstrationthat the immunoreactive kinin generatedduring this reaction represented a mixture of bradykinin and lysylbradykinin (206) suggested that more than one enzymewasinvolvedin kinin formationor conversion.Duringthe allergic response, both HMW and LMW kininogens enter nasal secretions by transudation from plasma(210). Since prekallikrein circulates in plasma as a complexwith HMW kininogen (12), it was not surprising that antibody that recognizes plasmaprekallikrein and kallikrein detected increased immunoreactive plasma(pre)kallikrein in nasal secretions during the allergic reaction (211). Biochemicalanalysis revealed, however,that prekallikrein activation occursduringthis process, suggestingthat plasma kallikrein contributes to the generation of bradykinin before subsequent inactivation by plasmaprotease inhibitors. Thepresence of complexesof C1inactivator with both kallikrein andHageman factor in nasal secretions during the immediateallergic response has recently been confirmed(D. Proud & A. P. Kaplan, unpublishedobservations) using specific assays (110, 111). Thus,it seemscertain that conversionof prekallikrein to kallikrein occursas a result of contact activation of Hageman factor either upona surface altered by initial inflammatory events or by somenegatively charged mucusmacromolecules or mast cell heparin (212). Althoughmast cell activation clearly occurs during the immediateallergic reaction and

Annual Reviews

Annu. Rev. Immunol. 1988.6:49-83. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

66

PROUD

& KAPLAN

tryptase can be detected in nasal secretions (211), the relative levels and activities of plasmakallikrein andtryptase wouldsuggest that the latter makes, at best, a minimalcontribution to bradykinin generation. Generation of lysylbradykinincannotbe explainedby the actions of either of these enzymes.This peptideis generatedby a tissue kallikrein that has been shown,for the first time, to be present in nasal secretions. Immunoreactive tissue kallikrein can be detected evenin resting nasal secretions, but concentrationsof this materialincreasein responseto antigenchallenge(213). Theidentity of this material as authentic tissue kallikrein wasconfirmed by physicochemical and immunological criteria as well as by its ability to generate lysylbradykinin. Althoughpresent in approximately10-fold lower concentrationsthan immunoreactive plasmakallikrein, tissue kallikrein is responsiblefor mostof the kininogenaseactivity in post-challengelavages (213), presumablydue to its resistance to inhibition relative to plasma kallikrein. This resistance to inhibition, together with a greater substrate availability, probablyexplains the fact that as muchas 45%of the kinin detected duringthis reaction represents lysylbradykinin(206, 214). Indeed, since postchallengenasal lavagesalso contain elevated levels of an aminopeptidase capable of converting lysylbradykininto bradykinin (215), the level of lysylbradykinin mayinitially be greater than 45%,emphasizing the importanceof tissue kallikrein in kinin-formationduringthe immediate allergic response. In addition to metabolismof lysylbradykinin by the aminopeptidase, both bradykinin and lysylbradykinin are metabolized by a carboxypeptidase N-like enzymewhich removes the C-terminal arginine from both peptides. Nasal lavages also contain low levels of angiotensin-converting enzyme.Althoughlittle, if any, bradykinin metabolismdue to angiotensin-converting enzymewas observed in lavages, this does not meanthat this enzymedoes not play a role in kinin metabolismin nasal secretions that have not been diluted by lavage. Thus, kinin metabolism in nasal secretions during the immediateallergic response showsseveral similarities to metabolismin plasmawhereinitial kinin hydrolysisis by carboxypeptidaseswith subsequentslower hydrolysis by angiotensin converting enzyme(96, 99). Interestingly, the actions of these peptidases nasal secretions could be expectedto lead to a temporalaccumulationof des (Arg9) bradykinin.This peptide is inactive in systemsregulated by the "classic" kinin receptor, called B2-typeby Regoliet al (97, 216)but is more active than bradykinin on the so-called B1receptor. Thus, dependingon 9) the type of kinin receptors in the nasal mucosa,formationof des (Arg bradykinincouldresult in terminationof biologicactivity (if B2receptors predominate)or the generation of a biologically active peptide (if receptors are present). This conceptis currently understudy.

Annual Reviews

Annu. Rev. Immunol. 1988.6:49-83. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

KININS

AND INFLAMMATION

67

In addition to the immediate allergic response, bradykinin and lysylbradykinin have also been shown to be generated during the late phase reaction to nasal challenge (217). The mechanismscontrolling kinin formation and metabolism during the late reaction have not yet been studied, but several differences betwten the immediate and late responses suggest this would be worthwhile. The profile of inflammatory mediators in the late reaction differs slightly from the immediateresponse. Whilehistamine is present in both reactions, prostaglandin D2 (PGD2), the major cyclooxygenase product of humanmast cells (218), is present in the early reaction but is undetectable during the late response. Since basophils makevery little PGD2(218), this suggests that this cell type mayparticipate in the late reaction. Also, while levels of histamine and TAME-esteraseactivity are similar during the immediateand late reactions, kinin levels are significantly lower during the late response (217). This mayreflect altered rates or pathways of formation associated, perhaps, with basophil rather than mast cell activation or different rates or mechanismsof kinin metabolism, particularly since inflammatorycells accumulate in the nasal mucosa during the late response (219). Kinin generation also occurs during the inflammatory response to nasal challenge with cold, dry air. Again, kinin formation is associated with a symptomatic response and with the production of a spectrum of inflammatory mediators (220, 221) virtually identical to that observed during the immediate allergic response. Although symptomsof rhinitis in response to cold, dry air challenge clearly involve mastcell activation, this activation is independent of antigen and seems to occur via different mechanism. Thus, while the tricyclic antihistamine, azatadine, is effective in blocking IgE-mediatedmast cell mediator release in vitro and in vivo (222), it does not prevent mediator release or symptomsin response to cold, dry air challenge (223). In each of the three types of rhinitis described above, kinin formation is associated with mast cell or basophil activation, and it would seem reasonable to assume that the release of mast cell/basophil mediators initiates the inflammatory response allowing influx of kininogens and plasmakallikrein to nasal secretions. The large circulating pools of plasma kininogens would provide a source for prolonged kinin formation which could maintain chronic symptomatology. The relationship between mast cell/basophil activation and kinin formation does not always hold, however. It is knownthat exposure of mast cells or basophils to hyperosmolar environments in vitro will induce mediator release (224). Nasal provocation with hyperosmolar mannitol similarly induces increased concentrations of histamine and leukotrienes in nasal secretions, but this mast cell activation is associated with little, if any, significant degree of kinin

Annual Reviews

Annu. Rev. Immunol. 1988.6:49-83. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

68

PROUD & KAPLAN

formation (225). Thus, kinin generation does not appear to be an automatic consequenceof mast cell activation. It is also possible to have symptomatic episodes of rhinitis in which kinin formation occurs independent of mast cell activation. In recent studies of induced rhinovirus colds, kinin generation did not occur in individuals who were infected but not symptomatic. During symptomatic infections, however, concentrations of kinins in lavages correlated with symptomscores, and the strength of this correlation increased with the severity of illness, i.e. the moresevere the cold the greater the relationship between the measurements of kinins and symptoms. In contrast to the various types of reactions described above, the mast cell mediators histamine and prostaglandin D2 were not increased during rhinovirus colds (226). Indeed, to date, kinins are the only mediators detected that correlate with the symptomatic response to rhinovirus infection. Despite the lack of mast cell activation during these infections, kinin generation occurs via mechanismssimilar to those delineated for the immediateallergic response. Both bradykinin and lysylbradykinin are produced, although it would appear that lysylbradykinin represents a lower proportion of the kinin generated than during the allergic reaction. In keeping with this, tissue kallikrein levels do not increase to the degree observed for the allergic response (D. Proud, unpublished observations). While the presence of kinins in nasal secretions during these various types of rhinitis suggests that these peptides maycontribute to the symptomatologyof rhinovirus colds as well as allergic reactions, such a role for kinins is clearly dependent upon their ability to induce relevant symptoms whenapplied to the nasal mucosa. It has recently been demonstrated that nasal provocation with bradykinin does, indeed, induce symptoms of nasal obstruction and rhinorrhea. Of particular relevance to the possible involvement of kinins in the commoncold is the observation that nasal provocation with bradykinin also leads to the development of a sore throat, presumably as a result of mucocilliary transport causing some kinin to reach pain receptors in the throat. The ability of bradykinin to induce symptomsis not mediated by mast cell activation (227). Thus, the ability of kinins to induce relevant symptomsin the nasal cavity together with the evidence that kinin generation occurs during various types of rhinitis provides strong support for the hypothesis that kinins may be important mediators of several types of inflammatory disorders of the upper airways. The recent developmentof the first competitive antagonists of bradykinin (228.) mayprovide a basis to establish definitively the extent to which kinins contribute to the symptomatology of such reactions.

Annual Reviews

Annu. Rev. Immunol. 1988.6:49-83. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

KININS AND INFLAMMATION 69

Asthma The edema-promoting and smoothmuscle-contractingproperties of kinins must also lead to consideration of whether these peptides participate in the pathogenesis of inflammatoryconditions of the lower airways, particularly asthma.Bolusintravenousinjections of bradykininhavebeen shownto induce transient respiratory distress in asthmatics but not in normalsubjects (229). Similarly, several inhalational studies have corroborated the ability of bradykinin to provokebronchospasmin human asthmatics but not in normalhumans(230, 232); this is consistent with the airways hyperreactivity typical of asthmatics (233). Early reports elevated systemic plasmakinins during asthmatic attacks (234, 235) provide somesupport for the involvementof kinins in asthma but, unfortunately, in light of current knowledgeregarding the problemsof measuring plasmakinins (208, 209), the accuracyof these early measurements must be considered dubious. Recent studies of bronchoalveolar lavage fluids, however,showedthat kininogenaseactivity could be detected in 22 of 24 samples from 17 asthmatics whoeither responded to aerosolized allergen challenge or had symptoms of active asthma, while such activity wasabsent in samplesfrom 6 of 7 normalcontrols (195). Levels of free kinins in lavagefluids correlatedwiththe presenceof kininogenase activity. The principal componentof this kininogenase was shownto be tissue kallikrein; in a small numberof samples,someplasmakallikrein activity wasalso present. Theprincipal control of this tissue kallikrein appearsto be by ~ 1-antiprotease(236). Thus,mechanisms for the generationof kinins are present and active duringasthma.Preliminaryevidencealso indicates elevatedtissue kallikrein andkinin levels in lavagefluids fromsubjectswith other types of local pulmonaryinflammation(bronchitis and pneumonitis) (195); this suggestsperhaps,in analogywith the upperairways,that kinins maybe involvedin a range of inflammatory disorders of the lowerairways. Hereditary Angioedema Hereditary angioedemais causedby low levels of functional C 1-INHdue to low total protein levels or synthesis of an abnormalC I-INH. In this disorder simultaneousactivation occurs of the Hageman factor-dependent pathwaysand the classical complementpathways. Patients present with episodic swelling occurring virtually anywherein the body, attacks of several abdominalpain due to edemaof the bowel wall, and laryngeal edema.Althougha kinin derived from a cleavage product of the second complementcomponentwas thought to be responsible for the severe swelling(237), other studies suggestbradykininto be the critical peptide

Annual Reviews

Annu. Rev. Immunol. 1988.6:49-83. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

70

PROUD & KAPLAN

(238, 239). Onthe other hand, a kinin-like vasoactive substancemayyet be shownto be a complementderivative, a point suggested by recent reports (240). Duringattacks of angioedemathere is depletion of prekallikrein and HMW kininogen(241), formation of bradykinin (242), whenblisters are inducedin the skin of patients, they are found to have elevated plasmakallikrein levels (243). Thecomplement activation appears due to autoactivation 0f C1(specifically Clr) in the absenceof CI-INH (244, 245). However,Hageman-factorfragment (but not HFa) has shownto activate enzymaticallythe first componentof complement (246) whenit is directly incubated with purified C1 or added to plasma. C1 activation is due to cleavage of the Clr subcomponent by HFf(247). hereditary angioedema,HFactivation maycontribute to the complement consumptionseen during episodes of swelling. Other Disorders Although we routinely use dextran sulfates as model compoundsupon which Hageman-factor activation can occur, other sulfated mucopolysaccharides maysubserve similar functions in vivo (248, 249). The synovial fluid of patients with rheumatoidarthritis contains plasmakallikrein, whichhas been shownto activate neutrophil procollagenase to collagenase(250). Uric acid and pyrophosphatecrystals can act as surfaces in contact activation (251,252)and maycontribute to the inflammationof gout and pseudo-gout.Thus, a role for the contact activation system in inflammatoryarthritides is possible, and this mayinvolveother functions of the enzymesformed,in addition to any effects of kinins. Activation of Hageman factor is also inducedby the Lipid A component of endotoxin (253-255), and prominentdepletion of contact activation proteins occurs in endotoxic shock (256-259). The pooling of fluid into bodycavities, the intravascular volumedepletion, and the hypotension seen maybe caused by release of bradykinin. Likewise, other bacterial infections such as typhoid fever have been shownto be associated with prekallikrein depletion, formation of kallikrein-C1 INHcomplexes,and CI-INHdepletion as evidence of contact activation (260). In patients hospitalized becauseof trauma, the onset of sepsis has beenshownto be associated with kininogendepletion and determinationof serial kininogen levels has prognostic value (257). Anunfortunate circumstancehas dramatizedthe kinin-formingcapacity of HFf; trauma patients given plasma protein fractions (as plasmaexpanders) that were contaminatedwith HFf suffered profoundhypotension(261,262). In patients with disseminatedintravascular coagulation due to endo-

Annual Reviews

Annu. Rev. Immunol. 1988.6:49-83. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

KININS

AND INFLAMMATION

71

thelial injury and/or endotoxemia(including gram-negativesepsis, grampositive sepsis, or viremia), decreasedplasmafactor XII, prekallikrein, and kallikrein inhibitory activity are seen (263). Thesechangeswerenot observedin disseminatedintravascular coagulation associated with leukemia, carcinoma, or abortion (264). These data suggest activation the Hagemanfactor~lependent pathways. A similar pattern of protein depletion has beenobservedin somepatients with polycythemia vera (265), type II hyperlipidemia(familial hypercholesterolemia)(266) and Rocky Mountainspotted fever (267). In somepatients with nephrotic syndrome, a similar diminutionof contact activation factors has beenreported (268) and this wasnot explicable simplyas a result of loss of proteins in the urine (269). Cirrhosis, as mightbe anticipated, is associated with diminished levels of plasmaprekallikrein, HMW-kininogen, and, to a lesser degree, Hageman factor, whichseem to be due to a diminished rate of protein synthesis(270).

SUMMARY Althoughconsiderable progress has been madein elucidating the molecular events occurring during kinin generation by both the plasmakininformingsystemandthe tissue kallikrein system,it is only in recent years that wehavecometo appreciate their potential role in inflammationin a wide variety of diseases. The importanceof the tissue kallikrein system dependsuponsecretion of the active form of the requisite enzymein the presence of a source of kininogen. Since tissue kallikreins are widely distributed in tissues, andsince lymphandinterstitial fluid containskininogen(271), a local milieu for potential kinin formationis alwayspresent. Theplasmasystemwill be activated secondaryto inflammationinitiated by someother process. There maybe endothelial or epithelial damage exposingconnectivetissue. Plasmaleakagecausedby release of someother permeabilityfactor (including kinin madeby tissue kallikrein) wouldthus lead to activation of the plasmacascadein manyformsof inflammation. As with all mediators,however,the contributionof kinins to an inflammatoryresponsecan only be definitively evaluatedif their actions can be selectively antagonized.Competitivereceptor antagonists have recently been synthesized (228) and will, we hope, soon be available for administration to humans.Shouldthese compounds proveeffective in vivo, they couldbe usedin conjunctionwith currentlyavailableassaysfor kallikreins, kininogens,kinins, and their various inactivated or degradedproducts, to provide newinsights into the role of these systemsin the pathogenesesof inflammatorydiseases.

Annual Reviews 72

PROUD & KAPLAN

ACKNOWLEDGMENTS The authors thank Bonnie Evans for excellent Proud acknowledges support from grant National Institutes of Health.

secretarial assistance. Dr. number HL32272 from the

Annu. Rev. Immunol. 1988.6:49-83. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

Literature Cited 1. Spragg,J., Talamo,R. C., Austen,K. F. 1970. Immunochemistryof bradykinin and immunologic activation of the kinin system. In Handbookof Experimental Pharmacolo#y:Bradykinin, Kallidin and Kallikrein, ed. E. G. Erdos, 25:531-49.Berlin: SpringerVerlag 2. Garcia-Leme, J. 1978. Bradykinin system. In Handbookof Experimental Pharmacology. Inflammation,ed. J. R. Vane, S. H. Ferreira, 50: 464-522. Berlin: Spdnger-Verlag 3. Marceau,F., Lussier, A., Regoli, D., Giroud, J. P. 1983. Pharmacologyof kinins: Their relevanceto tissue injury and inflammation.Gen. Pharmacol.14: 209-29 4. Kaplan,A. P., Silverberg, M.1987.The coagulation-kinin pathway of human plasma. Blood 70:1-15 5. Ratnoff, O. D., Rosenblum, J. M.1958. Role of Hageman factor in the initiation of clotting by glass: Evidence that glass frees Hageman factor from inhibition. Am. J. Med.25:16(~68 6. Wuepper,K. D. 1973. Prekallikrein deficiency in man. J. Exp. Med.138: 1345-55 7. Weiss,A. S., Gallin, J. I., Kaplan,A. P. 1974. Fletcher factor deficiency. Abnormalitiesof coagulation, fibrinolysis, chemotacticactivity, and kinin generation attributable to absence of prekallikrein.J. Clin. Invest. 53:622-33 8. Saito, H., Ratnoff, O. D., Donaldson, V. H. 1974.Defectiveactivationof clotting, fibrinolytie, and permeability enhancing system in humanFletcher trait plasma.Circ. Res. 34:641-51 9. Wuepper,K. D., Miller, D. R., LaCombe,M.J. 1975.Flaujeac trait. Deficiency of human plasma kininogen.J. Clin. Invest. 56:1663-72 10. Colman, R. W., Bagdasarian, A., Talamo,R. C., Scott, C. F., Seavey, M., Guimaraes,J. A., Pierce, J. V., Kaplan, A. P. 1975. Williamstrait: Humankininogen deficiency with diminishedlevels of plasminogenactivator and prekallikrein associatedwith

abnormalities of the Hagemanfactor dependentpathways.J. Clin. Invest. 56: 1650-62 11. Donaldson, V. H., Glueck, H. I., Miller, M.A., Movat,H. Z., Habal,F. 1976. Kininogendeficiency in Fitzgerald trait. Role of high molecular weightkininogenin clotting and fibrinolysis. J. Lab. Clin. Med.87:327-37 12. Mandle, R. Jr., Coleman, R. W., Kaplan, A. P. 1976. Identification of prekallikrein and HMW-kininogen as a circulating complex in human plasma.Proc.Natl, Acad.Sci. USA73: 4179-83 13. Thompson,R. E., Mandle, R. Jr., Kaplan,A. P. 1977. Associationof factor XI and high molecularweightkininogenin humanplasma.J. Clin. Invest. 60:1376-80 14. Ratnoff, O. D., Davie, J. W., Mallet, D. L. 1961. Studies on the action of Hagemanfactor. Evidence that activated Hageman factor in turn activates plasma thromboplastinantecedent. J. Clin. Invest. 40:803-19 15. Cochrane,C. G., Wuepper,K. D. 1971. Thefirst component of the kinin-forming system in human and rabbit plasma.Its relationshipto clotting factor XII (Hageman factor). J. Exp. Med. 134:986-1004 16. Kaplan, A. P., Austen, K. F. 1970. A prealbumin activator of prekallikrein. J. Immunol. 105:802-11 17. Kaplan, A. P., Austen, K. F. 1971. A prealbuminactivator of prekallikrein II. Derivation of activators of prekallikrein from activated Hageman factor with plasmin. J. Exp. Med.138: 696-712 18. Cochrane, C. G., Revak, S. D., Wuepper,K. D. 1973. Activation of Hagemanfactor in solid and fluid phase. J. Exp. Med. 138:1564-83 19. Hathaway,W. E., Belahusen, L. P., Hathaway,H. S. 1965. Evidence of a newplasmacoagulation factor I. Case report, coagulation studies and physiocochemical properties. Blood 26: 521-32

Annual Reviews

Annu. Rev. Immunol. 1988.6:49-83. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

KININS AND INFLAMMATION 20. Hattersley, P. G., Hayse,D. 1970.Fletcher factor deficiency.Areport of three unrelated cases. Br. J. Haematol.18: 411-16 21. Miller, G., Silverberg, M., Kaplan,A. P. 1980. Autoactivatability of human Hageman factor (factor XII). Biochem. Biophys. Res. Commun. 92:803-10 22. Silverberg,M., Dunn,J. T., Garen,L., Kaplan, A. P. 1980. Autoactivationof humanHagemanfactor. Demonstration utilizing a synthetic substrate. J. Biol. Chem.255:7281-86 23. Wiggins,R. C., Coehrane,C. G. 1979. Theautoactivation of rabbit Hageman factor. J. Exp. Med.150:1122-33 24. Tankersley, D. L., Finlayson, J. S. 1984.Kinetics of activation and autoactivation of humanfactor XII. Biochemistry 23:273-79 25. Tans,G., Rosing,J., Griffin, J. H.1983. Sulfatide-dependentautoactivation of humanblood coagulation factor XII (Hageman factor). J. Biol. Chem.258: 8215-22 26. Dunn,J. T., Silverberg,M., Kaplan,A. P. 1982.Thecleavageand formationof activated Hagemanfactor by autodigestion and by kallikrein. J. Biol. Chem.257:1779-84 27. McMillin,C. R., Saito, H., Ratnoff,O. D., Walton,A. G. 1974. Thesecondary structure of humanHagemanfactor (factor XII) andits alteration by activating agents.J. Clin. Invest. 54: 131222 28. Ratnoff, O. D., Saito, H. 1979. Amidolytic propertiesof singlechainactivated Hageman factor. Proc.Natl. Acad.Sci. USA76:1461-63 29. Heimark,R. L., Kurachi,K., Fujikawa, K., Davie, E. W.1980. Surface activation of coagulation, fibrinolysis, and kinin formation. Nature 286:456 -60 30. Kurachi, K., Fujikawa,K., Davie, E. W.1980. Mechanism of activation of bovinefactor XI by factor XII andfactor XIIa. Biochembtry19:1330--38 31. Silverberg, M., Kaplan, A. P. 1982. Enzymaticactivities of activated and zymogen forms of human Hageman factor (factor XII). Blood60:64-70 32. Morgan, P. H., Robinson, N. C., Walsh,K. A., Neurath, H. 1972. Inactivation of bovinetrypsinogenandchymotrypsinogenby diisopropyl phosphofluoridate. Proc. Natl. Acad. Sci. USA69:3312-16 33. Griffin, J. H. 1978.Roleof surface in surface-dependentactivation of Hagemanfactor (blood coagulation factor XII). Proc. NatL Acad. Sci. USA75:

73

1998-2002 34. Vander Graaf, F., Tans, G., Bouma, B. N., Griffin, J. H. 1982.Isolation and functional properties of the heavyand light chains of humanplasma kallikrein. J. Biol. Chem.257:14300-305 35. Rosing,J., Tans,G., Griffiti, J. H.1985. Surface-dependentactivation of human factor XII (Hageman factor) by kallikrein andits light chain. Eur. J. Biochem. 151:531-38 36. Webster,M.E., Pierce,J. V.1973.Activation of Hageman factor (factor XII). Identification andrelationship to kallikrein-kinin system.Fed. Proc.32:845 (Abstr.) 37. Schiffman, S., Lee, P. 1974. Preparation, characterization, and activation of a highlypurified factor XI. Evidence that a hitherto unrecognized plasmaactivity participatesin the interaction of factors XI and XII. Br. J. Haematol.27:101-14 38. Griffin, J. H., Cochrane,C. G. 1976. Mechanismsfor the involvement of high molecularweightkininogenin surface-dependent reactions of Hageman factor. Proc.Natl. Acad.Sci. USA73: 2554-58 39. Meier,H. L., Pierce, J. V., Colman, R. W., Kaplan,A. P. 1977. Activationand function of humanHagemanfactor. The role of high molecular weight kininogenand prekallikrein. J. Clin. Invest. 60:18-31 40. Wiggins, R. C., Bouma,B. N., Cochrane, C. G., Griffin, J. H. 1977. Role of high molecularweight kininogenin surface-binding and activation of coagulation factor XIa and prekallikrein. Proc. Natl. Acad.Sci. USA77: 4636-40 41. Silverberg, M., Nicoll, J. E., Kaplan, A. P. 1980. The mechanismby which the light chain of cleaved HMW-kininogen augmentsthe activation of prekallikrein, factor XI, andHageman factor. Thromb.Res. 20:173-89 42. Scott, C. F., Silver, L. D., Schapira, M., Colman,R. W. 1984. Cleavage of humanhigh molecular weight kininogen markedlyenhancesits coagulant activity. Evidencethat this molecule exists as a procofactor.J. Clin. Invest. 73:954-62 43. Bock, P. E., Shore, J. D., Tans, G., Gri~n,J. H. 1985.Protein-proteininteractions in contact activation of blood coagulation. Bindingof high molecular weightkininogenandthe 5-(iodoacetamido) fluorescein-labeled kininogen light chainto prekallikrein,kallikrein, and the separatedkallikrein heavyand

Annual Reviews

Annu. Rev. Immunol. 1988.6:49-83. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

74

PROUD & KAPLAN

light chains. J. Biol. Chem.260: 12434R. A., Colman,R. W., Schapira, M. 43 1984.Inactivation of factor XII active 44. Scott, C. F., Colman,R. W. 1980. fragment in normal plasma. PredomiFunction and immunochemistry ofprenantrole of Cl-inhibitor.J. Clin.Invest. kallikrein-high molccularweightkinin73:1542-49 ogencomplex in plasma.J. Clin. Invest. 56. Mandle,R. J. Jr., Kaplan,A. P. 1977. 63:413-21 Hagemanfactor substrates. I. Human 45. Cochrane, C. G., Revak, S. D. 1980. plasma prekallikrein. Mechanismof Disseminationof contact activation in activation by Hageman factor and parplasmaby plasma kallikrein. J. Exp. ticipation in Hagemanfactor-depenMed. 152:608-19 dent fibrinolysis. J. BioL Chem.252: 46. Revak,S. D., Cochrane,C. G., Griffin, 6097~5104 J. H. 1977. The binding and cleavage 57. Hojima,Y., Pierce, J. V., Pisano,J. J. characteristics of humanHageman fac1985.Purification andcharacterization tor during contact activation. A comof multiple formsof humanplasmaprekallikrein. J. Biol. Chem.260:400q5 parison of normalplasmawith plasmas deficient in factor XI, prekallikrein,or 58. Bouma,B. N., Miles, L. A., Barretta, high molecular weight kininogen. J. G., Griffin, J. H. 1980. Humanplasma Clin. Invest. 59:1167-75 prekallikrein. Studiesof its activation 47. Revak, S. D., Cochrane, C. G. 1976. byactivated factor XII and of its inacTherelationship of structure andfunctivation by diisopropyl phosphofluorition in humanHagemanfactor. The date. Biochemistry19:1151~50 association of enzymaticand binding 59. Bouma,B. N., Griffin, J. H. 1977. activities with separate regions of the Humanblood coagulation factor XI: Purification, properties, and mechmolecule.J. Clin. Invest. 57:852-60 48. Dunn,J. T., Kaplan,A. P. 1982. Foranismof activation by factor XII. J. mation and structure of humanHageBiol. Chem.252:6432-37 manfactor fragments.J. Clin. Invest. 60: Kurachi, K., Davie, E. W.1977. Acti7:627-31 vation of humanfactor XI (plasma 49. Burrowes,C. E., Movat,H. Z., Soltay, thromboplastinantecedent) by factor M. J. t975. The kinin systemof human XII (activated Hageman factor). Bioplasma, VI. The action of plasmin. chemistry 16:5831-39 Proc. Soc. Exp. Biol. Med. 135: 95961. Mannhalter, C., Schiffman, S. 1980. 66 Surface adsorption of factor XI. 50. McMullen,B. A., Fujikawa, K. 1985. Associationof adsorptionsites withthe Aminoacid sequence of the heavy heavy chain of activated factor XI. chain of humanc~-factor XIIa (actiThromb.Haemostasis43:124-26 vated Hageman factor). J. Biol. Chem. 62. Koide, T., Hermodson,M. A., Davie, 260:5328-41 E. W.1977.Activesite of bovinefactor XI (plasmathromboplastinantecedent). 51. Fujikawa, K., McMullen,B. A. 1983. Aminoacid sequenceof humanfl-facNature 266:729-30 tor XIIa. J. Biol. Chem.258:10924-33 63. Warn-Cramer, B. J., Bajaj, S. P. 1985. Stoichiometry of binding of high 52. Cool, D. E., Edgell, C. J. S., Lowie, G. V., Zoller, M. J., Brayer, G. O., molecular weight kininogento factor MacGillivray, R. T. A. 1985. CharXI/XIa. Biochem.Biophys. Res. Comacterization of humanblood coagumun. 133:417-22 lation factor XII cDNA.Prediction of 64. Thompson,R. E., Mandle, R. Jr., the primarystructure of factor XII and Kaplan, A. P. 1978. Characterization the tertiary structure of factor XIIa. J. of humanhigh molecularweight kininBiol. Chem.260:13666-76 ogen. Procoagulant activity associ53. Revak,S. D., Cochrane,C. G., Bouma, ated with the light chain of kinin-free B. N., Griffin, J. H. 1978.Surfaceand high molecular weight kininogen. J. fluid phase activities of two formsof Exp. Med. 147:488-99 activated Hagemanfactor produced 65. Nakayasu, T., Nagasawa, S. 1979. Studies on humankininogen. I. Isoladuringcontact activation of plasma.J. Exp. Med. 147:719-29 tion, characterization and cleavageby 54. Schreiber, A. D., Kaplan, A. P., plasma kallikrein of high molecular Austen, K. F. 1973. Inhibition by C1 weight (HMW) kininogen. J. Biochem. INHof Hageman factor fragment acti85:249-58 vation of coagulation,fibrinolysis, and 66. Keribiriou,D. M., Griffin, J. H. 1979. kinin generation. J. Clin. Invest. 52: High molecular weight kininogen. Studies of structure-functionrelation1402-9 ships and ofproteolysisof the molecule 55. DeAgostini,A., Lijnen, H. R., Pixley,

Annual Reviews

Annu. Rev. Immunol. 1988.6:49-83. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

KININS AND INFLAMMATION occurring during contact activation of plasma. J. Biol, Chem.254:12020-27 67. Mori, K., Nagasawa,S. 1981. Studies on human high molecular weight (HMW)kininogen II. Structural change of HMW-kininogenby the action of human plasmakallikrein II. J. Biochem. 84:1465-73 68. Komiya, M., Kato, H., Suzuki, T. 1972. Homologybetweenbovine high molecular weight and low molecular weight kininogen. Biochem.Biophys. Res, Commun.49:1438-43 69. Kitamura, N., Takagaki, Y., Furato, S., Tanaka,T., Nawa,H., Nakanishi, S. 1983. A single genefor bovinehigh molecular weight and low molecular weight kininogens. Nature305:545-49 70. Gournaris, A. D., Brown,M. A., Barrett, A. J. 1984. Humanplasma acysteine proteinase inhibitor. Purification by affinity chromatography, characterization, and isolation of an active fragment.Biochem.J. 221: 44552 71. Ohkubo,I., Kurachi, K., Takasawa, T., Shiokawa, H., Susaki,H. 1984. Isolation of a humaneDNAfor a2-Thiol proteinase inhibitor and its identity with low molecularweight kininogen. Biochemistry 23:5691-97 72. Muller-Esterl, W., Fritz, H., Machleidt, I. W., Ritonja, A., Brzin, J., Kotnik, M., Turk, V., Kellermann,J., Lottspeich, F. 1985. Humanplasma kininogensare identical with ~2-cysteine proteinase inhibitors. Evidence from immunological enzymological and sequence data. FEBSLett. 182: 310-14 73. Schmaier,A. H., Bradford,H., Farber, A., Silver, L. D., Schutsky,D., Scott, C. F., Colman, R. W. 1986. High molecular weight kininogen inhibits platelet calpain. J. Clin. Invest. 77: 1565-73 74. Thompson,R. E., Mandle, R. Jr., Kaplan,A. P. 1979.Studiesof the binding of prekallikrein and factor XI to high molecular weight kininogen and its light chain. Proc.Natl. Acad.Sci. USA76:4862~56 75. Keribiriou, D. M., Bouma,B. N., Griffin, J. H. 1980. Immunochemical studies of humanhigh molecularweight kininogenandits complexeswith plasmaprekallikreinor kallikrein. J. Biol. Chem. 255:3952-58 76. Bouma,B. N., Vlooswijk, R. A. A., Griffin, J. H. 1983.Immunologic studies of humancoagulationfactor XI and its complexwith high molecularweight kininogen. Blood 62:1123-31

75

77. Scott, C. F., Silver, L. D., Purdon,D., Colman, R, W. 1985. Cleavage of humanhigh molecular weight kininogenby factor XIa in vitro. Effect on structure and function. J. BioL Chem. 260:10856-63 78. Forbes,C. O., Pensky,J., Ratnoff, O. D. 1970. Inhibition of activated Hageman factor and activated plasma thromboplastinantecedent by purified C1inactivator. J. Lab. Clin. Med.76: 809-15 79. Pixley, R. A., Schapira, M., Colman, R. W.1985. The regulation of human factor XIIa by plasma proteinase inhibitors. J. Biol. Chem.260:1723-29 80. Harpel,P. C. 1976.Circulatoryinhibitors of humanplasma kallikrein. In ChemistryandBiologyof the KallikreinKininSystemin HealthandDisease,ed. J. J. Pisano, K. F. Austen, pp. 16977. DHEW Publ. No. (NIH) 76-791. Washington, DC 81. Gigli, I., Mason,J. W., Colman,R. W., Austen,K. F. 1970. Interaction of plasmakallikrein with C1inhibitor. J. Immunol. 104:574-81 82. McConnell,D. J. 1972. Inhibitors of kallikrein in humanplasma. J. Clin. Invest. 51:1611-23 83. Schapira, M., Scott, C. F., Colman,R. W.1982. Contribution of plasma protease inhibitors to the inactivation of kallikrein in plasma.J. Clin. Invest. 69: 46248 84. Van der Graaf, F., Koedam,J. A., Bouma,B. N. 1983. Inactivation ofkallikrein in plasma.J. Clin. Invest. 71: 149-58_ 85. Harpel, P. C., Lewin,M. F., Kaplan, A. P. 1985.Distribution of plasmakallikrein betweenC1 inactivator and macroglobulinin plasma utilizing a newassay for ~2-macroglobulin-kallikrein complexes.J. Biol. Chem.260: 4257-63 86. Heck,L. W., Kaplan,A. P. 1974. Substrates of Hageman factor: I. Isolation and characterization of humanfactor XI (PTA)and inhibition of the activated enzyme by ~ 1-antitrypsin. J. Exp. Med. 140:1615-30 87. Collen, D. 1976. Identification and someproperties of a new,fast reacting plasma inhibitor in humanplasma. Eur. J. Biochem.69:209-16 88. Scott, C. F., Schapira, M., James, H. L., Cohen,A. B., Colman,R. W.1982. Inactivation of factor XIa by plasma protease inhibitors. Predominantrole of ~ ~-proteaseinhibitor andprotective effect of high molecularweightkininogen.J. Clin. Invest. 69:844-52

Annual Reviews

Annu. Rev. Immunol. 1988.6:49-83. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

76

PROUD & KAPLAN

89. Moroi, M., Aoki, N, 1976. Isolation and characterization of ~t2-plasmin inhibitor from humanplasma.J. Biol. Chem. 351:5956~5 90. Mullertz, S., Clemmensen, J. 1976. The primaryinhibitor of plasminin human plasma. Biochem.J. 159:545-53 91. Koie, K., Ogata,K., Kamiya,T., Takamatsu,J. 1978. ~t plasmin-inhibitor deficiency (MiyasatoDisease). Lancet 2:1334-36 92. Ganrot, P. W.1967. Inhibition ofplasminactivity by ct2-macroglobulin. Clin. Chim. Acta 16:328-30 93. Schreiber, A. D., Kaplan, A. P., Austen, K. F. 1973. Plasmainhibitors of the componentsof the fibrinolytic pathwayin man. J. Clin. Invest. 52: 1394-1401 94. Harpel, P. C. 1977. Plasmininhibitor interactions. Theeffectivenessof ct2plasmininhibitor in the presenceof ~t 2macroglobulin. J. Exp. Med. 146: 1033-40 95. Erdos, E. G., Sloane, G. M. 1962. An enzymein humanplasma that inactivates bradykininand kallidins. Biochem. Pharmacol.11:585-92 96. Sheikh,I. A., Kaplan,A. P. 1986.Studies of the digestionof bradykinin,lysylbradykinin, and kinin degradation products by carboxypeptidase A, B, and N. Biochem.Pharmacol.35: 195763 97. Regoli, D., Barabe, S. 1980. Pharmacologyof bradykinin and related kinins. Pharmacol.Rev. 31:1-46 98. Sheikh,I. A., Kaplan,A. P. 1987. Evidence ofcarboxypeptidaseA and B like activities in the degradation of kininsin humanplasma and serum. Submitted 99. Sheikh,I. A., Kaplan,A. P. 1986.Studies of the digestionof bradykinin,lysylbradykinin,and des-arg9 bradykininby angiotensin converting enzyme.Biochem. Pharmacol.35:1951-56 100. Proctor, R. R., Rapaport, S. J. 1961. Thepartial thromboplastin time with kaolin:A simplescreeningtest for first stage dotting deficiencies. Am.J. Clin. Pathol. 35:212 101. Bouma, B. N., Kerbiriou, D. M., Vlooswijk,R. A. A., Griffin, J. H. 1980. Immunologicalstudies of prekallikrein, kallikrein, and high molecular weight kininogen in normal and deficient plasmasand in normalplasma after cold-dependent activation. J. Lab. Clin. Med. 96:693-709 102. Mancini, G., Carbonara, A. O., Heremans, J. F. 1965. Immunochemical quantificationof antigensby single radial immunodiffusion.Immunochemistry

2:235-54 103. Laurell, C. B. 1966.Quantitativeestimationof proteins by electrophoresis in agarose gel containing antibodies. Anal Biochem. 15:45-52 104. Tankersley,D. L., Alving, B. M., Finlayson,J. S. 1983.Activationof factor XII by Dextransulfate. Thebasis for an assay of factor XII. Blood62: 44856 105. Gallimore, M. J. 1983. Chromogenic substrate assays for studying componentsof the plasmakallikrein system in health and disease. Adv. Exp. Med. BioL 156A:225-31 106. Kluft, C. 1978. Determinationsofprekallikrein in humanplasma: Optimal conditions for activating prekallikrein. J. Lab. Clin. Med.91:83-95 107. Fisher, C. A., Schmaier,A. H., Addonizio, P., Colman,R. W.1982. Assay of prekallikrein in humanplasma: Comparisonof amidolytic, esterolytic, coagulationand imrnunochemical assays. Blood 59:963-70 108. DeAgostini,A., Schapira, M., Wachtfogel, Y. T., Colman,R. W.,Carrel, S. 1985. Human plasma kallikrein and CI inhibitor froma complexpossessingan epitope that is not detectable on the parent molecules:Demonstrationusing a monoclonalantibody. Proc. Natl. Acad. Sci. USA82:5190-93 109. Harpel,P. C. 1981.~t2-plasmininhibitor and ct2-macroglobulin-plasmin complexes in plasma. Quantitation by an enzyme-linked differential antibodyimmunosorbent assay. J. Clin. Invest. 68: 46-55 110. Kaplan, A. P., Gruber, B., lrlarpel, P. C. 1985. Assessment of Hageman factor activation in humanplasma. Quantification of activated Hageman factor-C1 inactivator complexesby an enzyme-linkeddifferential antibody immunosorbent assay. Blood 66:636-41 111. Lewin,M. F., Kaplan, A. P., Harpel, P. C. 1983. Studies of C1inactivatorplasmakallikrein complexesin purified systemsand in plasma.Quantification by enzyme-linked differential antibody immunosorbentassay. J, Biol. Chem. 258:6415-21 112. Shimamoto,K., Ando,T., Nakao, T., Tanaka, S., Sakuma, M., Miyahora, M. 1978. A sensitive radioimmunoassay method urinary kinins in man. J. Lab. Clin.forMed. 91:721-28 113. Odya,C. E., Moreland,P., Stewart, J. M., Parabe,J., Regoli, D. C. 1983. Developmentof a radioimmunoassay for des-Argg-bradykinin. Biochem.Pharmacol. 32:337-42

Annual Reviews

Annu. Rev. Immunol. 1988.6:49-83. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

KININS AND INFLAMMATION 77 114. Nustad, K., Orstavik, T. B., Gautvik, on the immunohistochemicallocalization of kallikrein in the pancreas.J. K. M., Pierce, J. V. 1978. Glandular kallikreins. Gen.Pharmacol.9:1-9 Histochem.Cytochem.29:985-88 115. Fiedler, F. 1979. Enzymologyofglandu-127. Amouric,M,, Lechenede la Porte, P., lar kallikreins. SeeRef. 1, 25(Suppl.): Figarella, C. 1982. Immunocytochemi103-61 ca1 localization of pancreatic kal116. Jacobsen, S. 1966. Separation of two likrein in humanacinar cells. Hoppedifferent substrates for plasmakininSeyler’s Z. Physiol. Chem.363:515-19 forming enzymes.Nature 210:98-99 128. Powers,C. A., Nasjletti, A. 1982. A novelkinin-generatingprotease(kinin117. Pierce, J. V., Guimaraes,J. A. 1976. Further characterization of highly puogenase)in the porcine anterior pituitary. J. Biol. Chem.257:5594-5600 rified humanplasma kininogens. See Ref. 80, pp. 121-27 129. Powers,C. A., Nasjletti, A. 1983. A 118. Nustad, K., Orstavik, T. B., Gautkininogenaseresemblingglandularkallikrein in the rat pituitary pars intervik, K. M. 1978. Radioimmunological measurements of rat submandibular media. Endocrinology112:1194-1200 gland kallikrein in tissues and serum. 130. Fuller,P. F., Clements, J. A., Whitfield, Microvasc.Res. 15:115-16 P. L., Funder,J. W.1985. Kallikrein 119. Lawton,W.J., Proud, D., Frech, M. geneexpressionin the rat anterior pituitary. Mol.Cell. Endocrinol.39:99-105 E., Pierce,J. V., Keiser,H. R., Pisano, J. J. 1981.Characterizationand origin 131. Pisano, J. J. 1975. Chemistry and ofimmunoreactive glandular kallikrein biologyof the kallikrein-kinin system. in rat plasma.Biochem.Pharmacol.30: In ProteasesandBiologicalControl,ed. 1731-37 E. Reich, D. B. Rifkin, E. Shaw,pp. 120. Rabito,S., Scicli, A. G., Kher,V., Car199-222. New York: Cold Spring retero, O. A. 1982. Immunoreactive Harbor Lab. glandular kallikrein in rat plasma:A 132. Proud,D., Bailey, G. S., Nustad,K., radioimmunoassay for its determinaGautvik, K. M. 1977. The immution. Am.J. Physiol. 242:H602-H610 nological similarity of rat glandular 121. Shimamoto,K., Mayfield, R. K., Markallikreins. Biochem.J. 167:835-38 golius, H. S., Chao, J., Stroud, W., 133. Fritz, H., Fiedler, F., Dietl, T., Kaplan, A. P. 1984. Immunoreactive Warwas,M., Truscheit, E., Kolb, H. tissue kallikrein in humanserum. J. J., Mair, G., Tschesche,H. 1977. On Lab. Clin. Med.103:731-38 the relationships betweenporcine pan122. Schachter, M., Peret, M.W., Billing, creatic, submandibular and urinary A. G., Wheeler, G. D. 1983. Immukallikreins. In Kininogenases 4. Physionolocalization of the protease kallogical Propertiesand Pharmacological likrein in the colon.J. Histochem. CytoRationale, ed. G. L. Haberland,J. W. Roher,T. Suzuki,pp. 15-28. Stuttgart/ chem. 31:1255-60 123. Lazure, C., Leduc,R., Seidah, N. G., NewYork: Schattauer-Verlag Chretien, M., Dube, J. Y., Chapde- 134. Fukushima, D., Kitamura, N., Naklaine, P., Frenette, G., Paquin, R., anishi, S. 1985. Nucleotidesequenceof Tremblay, R. R. 1984. The major cloned eDNAfor humanpancreatic androgen-dependentprotease in dog kallikrein. Biochemistry24:8037-43 prostate belongsto the kallikrein fam- 135. Baker, A. R., Shine, J. 1985. Human ily: Confirmation by partial aminoacid kidney kallikrein: eDNAcloning and sequencing. FEBSLett. 175:1-7 sequenceanalysis. DNA4:445-50 124. Pinkus,G.S., Maier,M., Seldin, D. C., 136. Mason,A. J., Evans,B. A., Cox,D. R., Ole-Moiyoi,O., Austen,K. F., Spragg, Shine,J., Richards,R. I. 1983.StrucJ. 1983. Immunohistochemical localture of a mousekallikrein gene family ization of glandular kallikrein in the suggestsa role in specific processingof endocrine and exocrine humanpanbiologically active peptides. Nature creas. J. Histochem. Cytochem. 31: 1279303:300-7 88 137. Richards, R. I., Catanzaro, D. F., 125. Dietl, T., Kruck,J., Fritz, H. 1978. Mason,A. J., Morris,B. J., Baxter,J. Localization of kallikrein in porcine D., Shine, J. 1982. Mouseglandular pancreas and submandibulargland as kallikrein genes. Nucleotidesequence revealed by the indirect immunofluofor cloned cDNAcoding for a member rescence technique. Hoppe-Seyler’s of the kallikrein arginyl esteropeptidase Z. Physiol. Chem.359:499-505 group of serine proteases. J. Biol. 126. Orstavik, T. B., Brandtzaeg, P., Chem. 257:2758-61 Nustad,K., Pierce, J. V. 1981.Effects 138. Ashley,P. J., MacDonald, R. J. 1985. of different tissue processingmethods Kallikrein-related mRNAs of the rat

Annual Reviews

Annu. Rev. Immunol. 1988.6:49-83. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

78

PROUD & KAPLAN

submaxillary gland: Nucleotide sequencesof four distinct types including tonin. Biochemistry24:4512-20 139. Fiedler, F., Werle,E. 1967. Twoprekallikreins from porcine pancreas. Hoppe-Seyler’sZ. Physiol. Chem.348: 1087-89 140. Matsas, R., Proud, D., Nustad, K., Bailey, G. S. 1981.Rapidpurification of a prekallikrein from rat pancreas. Anal. Biochem.113:264-70 141. Corthorn,J., Imanari,T., Yoshida,H., Kaizu,T., Pierce, J. V., Pisano, J. J. 1979. Isolation of prokallikrein from humanurine. Adv. Exp. Med. Biol. 120B:575-79 142. Brandtzaeg, P., Gautvik, K. M., Nustad,K., Pierce, J. V. 1976.Rat submandibulargland kallikreins: Purification andcellular localization.Br. J. Pharmacol.56:155-67 143. Fiedler, F., Hirschauer,C., Werle,E. 1970. Anreicherungvon prakallikrein B aus schweinepankreas and eigerschafter verschiedererformerdes pankreaskallikreins. Hoppe-Seyler’sZ. Physiol. Chem.351:225-38 144. Hojima, Y., Matsuda, Y., Moriwaki, C., Moriya,H. 1975. Homogeneity and multiplicity forms of glandular kallikreins. Chem.Pharmacol.Bull. 23: 1120-27 145. Schachter,M.1980.Kallikreins(kininogenases)--agroupof serine pro teases with bioregulatoryactions. Pharmacol. Rev. 31:1-17 146. Margolius,H. S., Horwitz,D., Geller, R. G., Alexander,R. W., Gill, J. R., Pisano,J. J., Keiser, H. R. 1974.Urinary kallikrein excretion in normal man.Relationships to sodium intake and sodium-retaining steroids. Circ. Res. 35:812-19 147. Vinci,J. M.,Zusman, R. M.,Izzo, J. L., Bowden, R. E., Horwitz,D., Pisano, J. J., Keiser, H. R. 1979. Human urinary and plasma kinins: Relationship to sodium-retaining steroids and plasma renin activity. Circ. Res. 44:22837 148. Margolius,H. S., Pisano,J. J., Geller, R., Sjoerdsma,A. 1971. Altered urinary kallikrein excretion in human hypertension. Lancet 2:1063-65 149. Horwitz,D., Proud, D., Lawton,W.J., Yates, K. N., Highet,P. F., Pisano, J. J., Keiser,H,R. 1982.Effectsof restriction of sodiumor administration of fludrocortisoneon parotid salivary kallikrein in man.J. Lab. Clin. Med.100: 146--54 150. Funder, J. W., Feldman,D., Edelman, I. S. 1972.Specific aldosteronebinding in rat kidney and parotid. J. Steroid

Biochem.3:209-18 151. Fuller, P. J., Funde~r,J. W.1986.The cellular physiologyof glandular kallikrein. KidneyInt. 29:953-64 152. Margolius,H. S., Chao,J., Kaizu,T. 1976. The effects of aldosterone and spironolactone on renal kallikrein. Clin. Sci. Mol.Med.51: 279s-82s 153. Chao, J., Margolius, H. S. 1983. Differentialeffects of testosterone,thyroxine and cortisol on rat submandibular glandvs renal kallikrein. Endocrinology 113:2221-25 154. Miller, D. H., Chao,J., Margolius,H. S. 1984.Tissuekallikrein synthesisand its modificationby testosterone or low dietary sodium.Biochem.J. 218:37-43 155. Powers,C. A., Nasjletti, A. 1984. A majorsex difference in kallikrein-like activity in the rat anterior pituitary. Endocrinology114:1841-44 156. Lieberthal, W., Oza,N. B., Arbeit, L., Bernard, D. B., Levinsky,N. G. 1983. Effects of alterations in sodiumand water metabolismon urinary excretion of active andinactive kallikrein in man. J. Clin. Endocrinol. Metab.56:513 19 157. Barton,S., Sanders,E. J., Schachter, M., Uddin, M. 1975. Autonomicnerve stimulation, kallikrein content and acinar cell granules of the cat’s submandibulargland. J. Physiol. 251: 36369 158. Orstavik, T. B., Gautvik, K. M. 1977. Regulationof salivary kallikrein secretion in the rat submandibulargland. Acta Physiol. Scand. 100:33-44 159. Farrell, M. M. 1960. Pancreatic kallikrein in physiological and pathological conditions. In Polypeptides Which Affect Smooth Muscles and BloodFessels, ed. M. Schachter, pp. 247-52. Oxford: Pergamon 160. Schachter,M., Peret, M. W., Billing, A. G., Wheeler,G. D., Longridge,D. J. 1983. Immunolocalization of the protease kallikrein in the colon. J. Histochem. Cytochem.31:1255-60 161. Derkx, F. H. M., Tan-Tjiong, H. L., Manin’t Veld, A. J., Schalekamp,M. P. A., Schalekamp, M. A. H. 1979. Activationof inactive plasmarenin by tissue kallikreins. J. Clin. EndocrinoL Metab. 49:765-69 162. Sealey,J. E., Atlas, S. A., Laragh,J. H., Oza, N. B., Ryan, J. W.1978. Human urinarykallikrein converts inactive to active renin and is a possible physiological activator of renin. Nature275: 144~45 163. Currie, M. G., Gellen, D. M., Chao,J., Margolius,H. S., Needleman,P. 1984. Kallikrein activation of a high molec-

Annual Reviews

Annu. Rev. Immunol. 1988.6:49-83. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

KININS AND INFLAMMATION ular weight atrial peptide. Biochem. Biophys. Res. Commun.120:46146 164. Thibault, G., Garcia, R., Cantin, M., Genest,J. 1984.Atrial natriuretic factor and urinary kallikrein in the rat: antagonistic factors? Can.J. Physiol. Pharmacol.62:645-49 165. Briggs, J., Marin-Grez, M., Steipe, B., Schubert, G., Schnermann,J. 1984. Inactivationof atrial natriuretic substance by kallikrein. Am.J. Physiol. 247:F480-F484 166. Cardin, A. D., Witt, K. R., Chat, J., Margolius, H. S., Donaldson,V. H., Hackson,R. L. 1984. Degradation of apolipoprotein B- 100 of humanplasma low density lipoproteins by tissue and plasmakallikreins. J. Biol. Chem.259: 8522-28 167. Proud, D., Knepper, M. A., Pisano, J. J. 1983. Distribution of immunoreactive kallikrein along the rat nephron. Am. J. Physiol. 244: F510~ F515 168. Omata,K., Carretero, O. A., Scicli, A. G., Jackson, B. A. 1982. Localization of active andinactivekallikrein (kininogenaseactivity) in the microdissected rabbit nephron.KidneyInt. 22:602-7 169. Tomita,K., Pisano,J. J. 1984. Binding of [3Hi bradykininin isolated nephron segmentsof the rabbit. Am.J. Physiol. 246:F732-F737 170. Schuster,V. L., Kokko,J. P., Jacobsen, H. R. 1984. Interactions of lysylbradykinin and antidiuretic hormone on the rabbit cortical collectingtubule. J. Clin. Invest, 73:1659-67 171. Tomita,K., Pisano, J. J., Knepper,M. A. 1985. Control of sodium and potassium transport in the cortical collecting duct of the rat. J. Clin. Invest. 76:132-36 172. Orstavik,T. B., Carretero, O. A., Scicli, A. G. 1982. Kallikrein-kininsystem in regulation of submandibular gland bloodflow. Am.J, Physiol. 242: H1010-H1014 173. Cuthbert, A. W., Margolius, H. S. 1982, Kinins stimulate net chloride secretionby the rat colon. Br. J. Pharmacol. 75:587-98 174. Musch,M. W., Kachur,J. F., Miller, R. J., Field, M., Stoff, J. S. 1983. Bradykinin-stimulated electrolyte secretion in rabbit andguinea-pigintestine. J. Clin. lnvest. 71:1073-83 175. Moriwaki,C., Fujimori,H. 1981.Studies on kallikreins. VI. Effect of pancreatic kallikrein and kinin on the transport of aminoacids and glucose across the rat small intestine. Chem. Pharmacol.Bull. 29:804--9

79

176. Mindroiu,T., Scicli, G., Perini, F., Carretero, O. A., Scicli, A. G.1986.Identification a new kinin in humanurine. J. Biol. of Chem. 261:7407-11 177. Webster, M. E., Mares-Guia, M., Diniz, C. R. 1970. Kallikreins in glandulartissues. SeeRef. 1, pp. 13155 178. Iwanaga, S., Han, Y. N., Kato, H., Suzuki,T. 1977.Actionsof variouskallikreins on HWM kininogen and its derivatives. SeeRef. 133,pp. 79-90 179. Geiger, R., Stuckstedte, U., Clausnitzer, B., Fritz, H. 1981.Progressive inhibition of humanglandular (urinary) kallikrein by humanserumand identification of the progressiveantikallikreinas ~ t-antitrypsin(~ ~-protease inhibitor). Hoppe-Seyler’s Z. Physiol. Chem. 362:317-25 180. Chat, J., Tanaka,S., Margolius,H. A. 1983.Inhibitory effects of sodiumand other monovalentcations on purified versus membrane-bound kallikrein. J. Biol. Chem.258:6461-65 181. Lieberthal, W.,Oza,N. B., Bernard,D. B., Levinsky,N. G. 1982.Theeffect of cations on the activity of humanurinary kallikrein. J. Biol. Chem.257: 10827-830 182. Newball,H. H., Talamo,R. C., Lichtenstein, L. M. 1979. Anaphylactic release of a basophil kallikrein-like activity. II. A mediatorof immediate hypersensitivity reactions. J. Clin. Invest. 64:466-75 183. Newball, H. H., Berninger, R. W., Talamo, R. C., Lichtenstein, L. M. 1979. Anaphylacticrelease of a bastphil kallikrein-like activity. I. Purification and characterization. J. Clin. Invest. 64:457~55 184. Schwartz,L. B., Ceidel, C., Caulfield, J. P., Wasserman, S. I., Austen,K. F. 1981, Cell association of complexesof chymase, heparin proteoglycan, and protein after degranulationby rat mast cells. J. Immunol.126:2071-76 185. Wintroub, B. U., Kaempfer, C. E., Schechter, N. M., Proud, D. 1986. A humanlung mast cell chymotrypsinlike enzyme.Identification and partial characterization. J. Clin. Invest. 77: 19 6-201 186. Meier, H. L., Newball, H. H., Berninger, R. W., Talamo,R. C., Lichtenstein, L. M. 1979. Purification of lung kallikrein of anaphylaxis. J. Aller#y Clin Immunol.63:191(Abstr.) 187. Meier, H. L., Kaplan, A. P., Lichtenstein, L. M., Revak,S., Cochrane,C. G., Newball,H. H. 1983. Anaphylactic release ofa prekallikreinactivator from

Annual Reviews

Annu. Rev. Immunol. 1988.6:49-83. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

80

PROUD & KAPLAN

human lungin vitro. J. Clin. Invest. 72: 574-82 188. Meier,H. L., Flowers,B., Silverberg, M., Kaplan, A. P., Newball, H. H. 1986. TheIgE-dependentrelease of a Hagemanfactor cleaving factor from humanlung. Am.J. Pathol. 123: 14654 189. Newball,H. H., Meier,H. L., Kaplan, A. P., Revak,S. D., Cochrane,C. G., Lichtenstein,L. M. 1981.Activationof Hageman factor by proteases released during antigen challenge of human lung. Trans. Assoc. Am.Phys. 94: 12634 190. Meier,H. L., Flowers,B., Silverberg, M., Kaplan, A. P., MaeGlashan, D. W. Jr., Schulman,E. S., Newball,H. H. 1982. Localization and characterization of lung Hageman factor activator. Fed. Proc.41:377(Abstr.) 191. Proud, D., MacGlashan,D. W. Jr., Newball,H. H., Schulman,E. S., Lichtenstein, L. M. 1985. Immunoglobulin E-mediatedrelease of a kininogenase from purified humanlung mast cells. Am.Rev. Respir. Dis. 132:405-8 192. Maier,M., Spragg,J., Schwartz,L. B. 1983. Inactivation of human high molecular weight kininogen by human mast cell tryptase. J. Immunol.130: 2352-56 193. Schwartz,L. B., Maier,M., Spragg,J. 1986. Interaction of humanlow molecular weight kininogen by humanmast cell tryptase. Adv. Exp. Med. Biol. 198A:105-11 194. Proud,D., Siekerski,E. S., Bailey, G. S. 1987. Human lung mastcell tryptase is a kininogenase. Fed. Proc. 46:932 (Abstr.) 195. Christiansen, S. C., Proud, D., Cochrane, C. G. 1987. Detectionof tissue kallikrein in the bronchoalveolarlavagefluids of asthmaticsubjects.J. Clin. Invest. 79:188-97 196. Waxman, L. 1981. Calcium-activated proteases in mammalian tissues. Methods Enzymol. 80:664-80 197. Knauer, K. A., Lichtenstein, L. M., Adkinson,N. F., Fish, J. E. 1981. Platelet activation during antigen-induced airwayreactions in asthmaticsubjects. N. En#l. J. Med. 304:1404-07 198. Higashiyama,S., Ishiguro, H., Ohkulso, I., Fujimoto,S., Matsuda,T., Sasaki, M. 1986. Kinin release from kininogensby calpains. Life Sci. 39: 1639-44 199. Sueyoshi,T., Enjyoji, K., Shimada,T., Kato, H., Iwanaga, S., Bando, Y., Kominami,E., Katunuma,N. 1985. A newfunction of kininogens as thiol

proteinaseinhibitors: Inhibitionof papain and cathepsins B, H and L by bovine, rat and humanplasma kininogens. FEBSLett. 182:193-95 200. Melmon,K. L., Cline, M. J. 1967. Interactions of plasmakinins andgranulocytes. Nature213:90-92 201. Movat,H. Z., Steinberg,G. S., Habal, F. M., Ranadive, N. S. 1973. Demonstration of a kinin-generating enzymein the lysosomesof humanpolymorphonuclear leukocytes. Lab. Invest. 29:669-84 202. Wasi,S., Movat,H. Z., Pass, E., Chan, J. Y. C. 1978. Production, conversion and destruction of kinins by human neutrophilleukocyteproteases. In Neutral Proteases of HumanPolymorphonuclear Leukocytes, ed. K. Havemann, A. Janoff, pp. 245~0. Baltimore/ Munich: Urban & Schwarzenburg 203. Lupke,U., Rautenberg,W., Tschesche, H. 1982. Kininogenase from human polymorphonuclearleukocytes. Agents Actions (Suppl.) 9:308-14 204. Greenbaum, L. M. 1979. Kininogenasesof blood cells (alternate kinin generating systems). See Ref. 1, 25 (Suppl.): 91-102 205. Naclerio, R. M., Meier, H. L., KageySobotka, A., Adkinson, N. F. Jr., Meyers,D. A., Norman,P. S., Lichtenstein, L. M. 1983. Mediatorrelease after nasal airwaychallengewithallergen. Am.Rev. Respir Dis. 128:597-602 206. Proud,D., Togias,A., Naclerio, R. M., Crush, S. A., Norman,P. S., Lichtenstein, L. M. 1983. Kinins are generated in vivo followingnasal airway challenge of allergic individuals with allergen. J. Clin. Invest. 72:1678-85 207. Creticos,P. S., Peters, S. P., Adkinson, N. F. Jr., Naclerio, R. M., Hayes,E. C., Norman,P. S., Lichtenstein, L. M. 1984.Peptideleukotriene release after antigen challengein patients sensitive to ragweed.N. Engl. J. Med.310: 162630 208. Hulthen,U. L., Borge,T. 1976. Determination of bradykinin in blood by a sensitive radioimmunoassay. Scand.J. Clin. Lab. Invest. 36:833-39 209. Scicli, A. G., Mindroiu,T., Scicli, G., Carretero, O. A. 1982. Bloodkinins, their concentrationin normalsubjects and in patients with congenital deficiency in plasma kallikrein and kininogen.J. Lab. Clin. Med.100: 8193 210. Baumgarten, C. R., Togias, A. G., Naclerio, R. M., Lichtenstein, L. M., Norman,P. S., Proud, D. 1985. Influx of kininogens into nasal secretionsafter

Annual Reviews

Annu. Rev. Immunol. 1988.6:49-83. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

KININS AND INFLAMMATION

81

antigen challengeof allergic individmastcell involvement.J. Clin. lnvest. 76:1374-81 uals. J. Clin. lnvest. 76:191-97 211. Baumgarten,C. R., Nichols, R. C., 221. Togias,A. G., Naclerio, R. M., Peters, Naclerio, R. M., Liehtenstein, L. M., S. P., Nimmagadda,I., Proud, D., Norman,P. S., Proud, D. 1986. PlasKagey-Sobotka,A., Adkinson, N. F. Jr., Norman, P. S., Lichtenstein,L. M. makallikrein during experimentallyinducedallergic rhinitis: Rolein kinin 1986. Local generation of sulfidopeptide leukotrienesuponnasal provoformation and contribution to TAMEcation with cold, dry air. Am. Rev. esterase activity in nasal secretions.J. lmmunol. 137:977-82 Respir. Dis. 133:1133-37 212. Hojima,Y., Cochrane,C. G., Wiggins, 222. Togias, A. G., Naclerio, R. M., Warner, J., Proud, D., Nimmagadda, R. C., Austen, K. F., Stevens, R. L. 1984.In vitro activation of the contact I., Norman,P. S., Lich~enstein,L. M. (Hageman factor) system of plasma 1986. Demonstrationof inhibition of heparin and chondroitin sulfate E. mediatorrelease fromhumanmastcells by azatadinebase: In vivo andin vitro Blood 63:1453-59 213. Baumgarten,C. R., Nichols, R. C., evaluation. J. Am. Med. Assoc. 255: Naclerio, R. M., Proud, D. 1986. Con225-29 centrations of glandular kallikrein in 223. Togias, A. G., Proud, D., Kageyhumannasal secretions increase during Sobotka,A., Norman,P., Lichtenstein, experimentally-induced allergic rhinitis. L., Naclerio, R. 1987. Theeffect of a J. lmmunol.137:1323-28 topical tricyclic antihistamineon the Proud, D., Baumgarten, C. R., response of the nasal mucosato chal214. Naclerio, R. M., Lichtenstein, L. M. lenge withcold, dry air and histamine. J. Allergy Clin. Immunol.79:599-604 1986. The role of kiains in human allergic disease. N. Engl. Reg. Allergy 224. Eggleston, P. A., Kagey-Sobotka,A., Proc. 7:213-18 Schleimer,R. P., Lichtenstein, L. M. 215. Proud, D., Baumgarten, C. R., 1983. Interaction betweenhyperosmolar and IgE-mediated histamine Naclerio, R. M., Ward, P. E. 1987. Kinin metabolismin nasal secretions release frombasophilsand mastcells. during experimentally-induced allergic Am.Rev. Respir. Dis. 130:86-91 rhinitis. J. Imtnunol.138:428-34 225. Silber, G., Naclerio,R., Eggleston,P., 216. Regoli, D., Marceau,F., Barabe, J. Proud,D., Togias,A., Lichtenstein,L. 1978. De novo formation of vascular M., Norman,P. 1985. In vivo release receptors for bradykinin. Can. J. of histamineby hyperosmolarstimuli. J, Allergy Clin. Immunol. 75: 176 Physiol. Pharmacol.56:674-77 217. Naclerio, R. M., Proud,D., Togias, A. (Abstr.) G., Adkinson,N. F. Jr., Meyers,D. A., 226. Naclerio, R. M., Gwaltney,J. M., HenKagey-Sobotka,A., Plant, M., Nordley, J. O., Eggleston,P., Baumgarten, man,P. S., Lichtenstein, L. M.1985. C. R., Lichtenstein, L. M., Proud, D. Inflammatorymediators in late anti1985. Kininsare generatedduring rhigen-inducedrhinitis. N. Engl. J. Med. novirus colds. Clin. Res. 33: 613A 313:65-70 (Abstr.) 218. MacGlashan, D. W.Jr., Schleimer, R. 227. Proud, D., Reynolds, C. J., LaCapra, P., Peters, S. P., Schulman,E. S., S., Kagey-Sobotka,A., Lichtenstein, Adams,G. K. III, Kagey-Sobotka,A., L. M., Naclerio, R. M. 1987. The Newball, H. H., Lichtenstein, L. M. responseto nasal provocationwith bra1983. Comparativestudies of human dykinin.J. Allergy Clin. Immunol.79: basophilsandmastcells. Fed. Proc.42: 254(Abstr.) 2504-9 228. Vavrek,R. J., Stewart, J. M. 1985. 219. Bascom,R., Pipkorn,U., Gleich, G. J., Competitiveantagonists of bradykinin. Lichtenstein, L. M., Naclerio, R. M. Peptides 6:161~4 1986. Effect of systemic steroids on 229. Newball,H. H., Keiser, H. R., Pisano, eosinophils (EOS)and majorbasic proJ. J. 1975. Bradykininand humanairtein (MBP)during nasal antigen chalways.Respir. Physiol. 24:139-46 lenge. J. Allergy Clin. Imrnunol.77 230. Herxheimer,H., Stresemann,E. 1961. (Suppl.): 246(Abstr.) Theeffect of bradykinin aerosol in 220. Togias,A. G., Naclerio, R. M., Proud, guineapigs and man.J. Physiol. London 158:38-39 D., Fish, J. E., Adkinson,N. F. Jr., Kagey-Sobotka, A., Norman,P. S., 231. Varonier,H. S., Panzani,R. 1968.The Lichtenstein, L. M. 1985. Nasal chaleffect of inhalation of bradykininon lengewithcold,dryair results in release healthy and atopic (asthmatic) chilof inflammatorymediators. Possible dren. Int. Arch.Allergy 34:293-96

Annual Reviews

Annu. Rev. Immunol. 1988.6:49-83. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

82

PROUD & KAPLAN

232. Fuller, R. W.,Dixon,C. M.S., Dollery, C. T., Barnes,P. J. 1986.Prostaglandin D2potentiates airwayresponsivenessto histamine and methacholine.Am.Rev. Respir. Dis. 133:252-54 233. Orehek,J., Gayrard,P., Smith,A. P., Grimaud,C., Charpin, J. 1977. Airway response to catbachol in normal and asthmatic subjects. Am. Rev. Respir. Dis. 115:937-43 234. Abe, K., Watanabe,N., Kumagai,N., Mouri, T., Seki, T., Yoshinaga, K. 1967. Circulating plasmakinin in patients with bronchial asthma.Experientia 23:626-27 235. Lukjan,H., Hofman,J., Kiersnoroska, B., Bielawiec, M., Chyrek-Borowska, S. 1972. The kinin systemin allergic states. Allerg. Irnmunol.Band.18: 2530 236. Christiansen, S. C., Proud,D., Zuraw, B. L., Cochrane,C. G. 1987.Inhibition of bronchialkallikrein by alpha- 1-proteinase inhibitor (~-I-PI) in asthma. Aller#y Clin. Immunol.79:257(Abstr.) 237. Donaldson,V. H., Rosen,F. S., Bing, D. H. 1977. Role of the second component of complement(C2) and plasrain in kinin release in hereditary angioneurotic adema(H.A.N.E.) plasma. Trans. Assoc. Am. Physicians 40: 174-83 238. Fields, T., Ghebrehiwet, B., Kaplan,A. P. 1983.Kinin formationin hereditary angioedemaplasma: Evidence against kinin derivation from C2 and in support of "spontaneous" formation of bradykinin. J. Allergy Clin. Immunol. 72:54~50 239. Curd,J. G., Yelvington,M., Burridge, N., Stimler,N. P., Gerard,C., Prograis, L. J. Jr., Cochrane,C. G., Muller-Eberhard, H. J. 1983. Generationof bradykinin during incubation of hereditary angioedemaplasma. Mol. lmmunol. 19:1365(Abstr.) 240. Strang, C. J., Auerbach,H. S., Rosen, F. S. 1986. Cls-inducedvascular permeabilityin C2-deficientguineapigs. J. Immunol. 137:631-35 241. Schapira,M., Silver, L. D., Scott, C. F., Schmaier,A.H., Prograis,L. J. Jr., Curd, J. G., Colman,R. W.1983. Prekallikrein activation and high-molecular-weight kininogenconsumption in hereditary angioedema. N. Engl. J. Med. 308:1050-53 242. Talamo,R. C., Haber, E., Austen, K. F. 1969. A radioimmunoassay for bradykininin plasmaandsynovialfluid. J. Lab. Clin, Med. 74:816-27 243. Curd,J. G., Prograis, L. F. Jr., Cochrane, C. G. 1980. Detection of active

kallikrein in inducedblister fluids of hereditary angioedemapatients. J. Exp. Med. 152:742-47 244. Ziccardi, R. J. 1982.Spontaneous activation of the first component of human complement (C1) by an intra molecular autocatalytic mechanism.J. lmrnunol. 128:2500-4 245. Cooper, N. R. 1983. Activation and regulation of the first componentof complement. Fed. Proc. 42: 134 (Abstr.) 246. Ghebrehiwet, B., Silverberg, M., Kaplan, A. P. 1981. Activation of the classical pathway of complementby Hagemanfactor fragment. J. Exp. Med. 153:665-76 247. Ghebrehiwet, B., Randazzo, B. P., Dunn,J. T., Silverberg,M., Kaplan,A. P. 1983. Mechanismof activation of the classical pathwayof complement by Hageman factor fragment. J. Clin. lnvest. 71:1450-56 248. Moskowitz,R. W., Schwartz, H. J., Michel,B., Ratnoff, O. D., Astrup, T. 1970. Generationof kinin-like agents by chrondroitinsulfate, heparin, chitin sulfate, andhumanarticular cartilage: Possible pathophysiologic implications. J. Lab. Clin. Med.76:790-98 249. Silverberg, M., Diehl, S. V. 1985. Autoactivation of humanfactor XII (Hagemanfactor): Effect of heparin and low molecularweight dextran sulfate. Blood 66(Suppl. I. No. 5): 343 (Abstr.) 250. Nagase,H., Cawston,J. E., DeSilva, M., Barrett, A. J. 1982. Identification of plasmakallikrein as an activator of latent collagenase in rheumatoid synovial fluid. Biochem.Biophys.Acta 707:133-42 251. Kellermeyer,R. W., Breckenridge,R. T. 1965. The inflammatoryprocess in acute goutyarthritis. I. Activationof Hageman factor by sodiumurate crystals. J. Lab. Clin. Med.63:307-15 252. Ginsberg,M., Jaques, B., Cochrane,C. G., Griffin, J. H. 1980.Uratecrystaldependentcleavage of Hageman factor in humanplasmaand synovial fluid. J. Lab. Clin. Med. 95:497-506 253. Morrison,D. C., Cochrane,C. G. 1974. Direct evidence for Hagemanfactor (factor XII) activation by bacterial lipopolysaccharides(endotoxins.). Exp. Med. 140:797-811 254. Pettinger, M. A., Young, R. 1970. Endotoxin-inducedkinin (bradykinin) formation: Activation of Hageman factor plasmakallikrein in humanplasma. Life Sci. 9:313-22 255. Kimball,H. R., Melmon, K. L., Wolff,

Annual Reviews

Annu. Rev. Immunol. 1988.6:49-83. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

KIN1NS AND INFLAMMATION S, M, 1972. Endotoxin-induced kinin production in man. Proc. Soc. Exp. BioL Med. 139:1078-82 256. Mason,J. W,, Kleeberg, U. R., Dolan, P, L., Colman,R. W. 1970. Plasma kallikrein and Hagemanfactor in gramnegative bacteremia. Ann. Int. Med. 73: 545-51 257. Hirsch, E. F., Nagajima, T., Oshima, G., Erdos, E. G., Herman, M. 1974. Kinin-sy.stem responses in sepsis after trauma m man. J. Sur#. Res. 17: 14753 258. Robinson, J. A, Klodynicky, M. L., Loeb, H. S., Recic, M. R., Gunnar, R. M. 1975, Endotoxin, prekallikrein, complement, and system vascular resistances. Sequential measurementin man. Am. J. Med. 59:61-67 259. O’Donnell, T. F., Clowes, G. H. Jr., Talarao, R. C., Colman, R. W. 1976. Kinin activation in the blood of patients with sepsis. Sury. Gynecol. Obstet. 143:539--45 260. Colman, R. W., Edelman, R., Scott, C. F., Gihnan, R. H. 1978. Plasma kallikrein activation and inhibition during typhoid fever. & Clin. Invest. 61: 287261. Alving, B. M., Hojima, Y., Pisano, J. J., Mason, B. L., Buckingham, R. E. Jr., Mozen, M. M., Finlayson, J. S. 1978. Hypotensionassociated with prekallikrein activator (Hagemanfactor fragments) in plasma protein fraction. N. Engl. J. Med. 299:66-70 262. Alving, B. H., Tankersley, D. L., Mason, B, L., Rossi, F., Aronson, D. L., Finlayson, J. S. 1980. Contact activation factors: Contaminants ofimmunoglobulin preparations with coagulant and vasoactive properties. J. Lab. Clin. Med. 96:33zl-46

83

263. Mason, J. W., Colman, R. W. 1971. The role of Hageman factor in disseminated intravascular coagulation induced by sepsis, neoplasia, or liver disease. Thromb. Diath. Haemorrh. 26: 325-31 264. Minna, J, D., Robboy, S. J., Colman, R.W. 1974. Disseminatedlntravascular Coagulation in Man, pp. 70-76. Springfield, I11: Charles IL Thomas 265. Carvalho, A, C., Ellman, L. 1976. Activation of the coagulation system in polycythemia vera. Blood 47:669-78 266. Carvalho, A. C., Lees, R. S., Vaillancourt, R. A., Cabral, R. B,, Colman, R. W. 1978. Activation of the kallikrein system in hyperbetalipoproteinemia. J. Lab. Clin. Med. 91:117-22 267. Yamada,J., Herber, P., Pettit, G. W., Wing, D. A., Oster, C. N. 1978. Activation of the kallikrein-kinin system in Rocky Mountain Spotted Fever. Ann. lnt. Med. 88:764~68 268. Honig, G. R., Lindley, A. 1971. Deficiency of Hagemanfactor (factor XII) in patients with the nephrotic syndrome. J. Pediatr, 78:633-37 269. Lange, L. G. III, Carvalho, A., Bagdasarian, A., Lahiri, B., Colman,R. W, 1974. Activation of Hagemanfactor in the nephrotic syndrome. Am. J. Med. 56:56549 270. Wong, P., Colman, R. W., Talamo, R. C., Babior, B. 1972. Kallikrein-bradykinin system in chronic alcoholic liver disease. Ann. Int. Med. 77:205-9 271. Proud, D., Nakafnura, S., Carone, F. A., Herring, P. L., Kawamura, M., Inagami, T., Pisano, J. J. 1984. The kallikrein-kinin system and renin-an. giotension system in rat renal lymph. Kidney Int. 25:880-85

Annu. Rev. Immunol. 1988.6:49-83. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

Annu. Rev. Immunol. 1988.6:49-83. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

Annual Reviews www.annualreviews.org/aronline

Annu. Rev. Immunol. 1988.6:85-113. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Ann. Rev. ImmunoL1988. 6:85-113 Copyright©1988by AnnualReviewsInc. All rights reserved

IMMUNOBIOLOGYOF CR2, THE B LYMPHOCYTE RECEPTOR FOR EPSTEIN-BARR VIRUS AND THE C3d COMPLEMENT FRAGMENT Neil R. Cooper, Margaret D. Moore, and Glen R. Nemerow ResearchInstitute of Scripps Clinic, Departmentof Immunology, 10666North Torrey Pines Road, La Jolla, California 92037 INTRODUCTION The complementsystem is the primary mediator of the biological consequencesof antigen-antibodyreactions. It consists of at least 20 chemically and immunologicallydistinct plasma and cell membraneproteins which acquire the ability to interact with one another, with antibody, and with cell membranes after activation of the system(1, 2). Theseinteractions directly generate the various biologic activities that complement mediates andwhichrangefromlysis of different kindsof cells, bacteria, andviruses to direct mediationof inflammatoryprocesses. In addition, complement is able to enlist the participation of other humoraland cellular effector systemsandto inducehistaminerelease frommastcells, directed migration of leukocytes, phagocytosis, and release of lysosomalconstituents from phagocytes. Complement also possesses immunoregulatoryactions which are, as yet, poorlyunderstood. Withthe exceptionof its membrane disrupting, cytolytic functions, all of the biological activities of the complement system~re mediatedthrough complementreceptors. Complement receptors are cell surface structures that specifically interact with complement protein cleavageproductsproducedduring the activation process(2-4). There are one or moredistinct complement receptors on the surface of mosttypes of circulating cells and 85 0732-0582/88/0410-0085502.00

Annual Reviews

Annu. Rev. Immunol. 1988.6:85-113. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

86

COOPER,

MOORE & NEMEROW

on manytissue cells. Interactions of the complementligands with these specific cellular receptors trigger various responses including cellular activation,, secretion of mediators, ingestion, directed migration, or other activities, depending on the receptor engagedand the cell type. Of the complement factors, the third component of complement, C3, plays a central role in the complementreaction sequence as it is activated as a consequenceof triggering of the complementsystem by either of the two activation pathwaysand because it is a key constituent of both of the enzymesthat mediate assemblyof the terminal, cytolytically active, portion of the complementreaction sequence. C3 also possesses multiple biological activities mediated by binding of the six distinct C3 activation/processing fragments to specific C3 receptors on the surface of various types of cells. Twoof these receptors, CR1and CR2, are present on the plasma membraneof lymphocytes (3, 4). CR1, found on most B cells, a minor proportion of T cells, and manyother cell types, is a single chain glycoprotein which exists in several allotypic forms varying between 160 and 250 kd. CR1interacts preferentially with the initial activation/processing products of C3 and C4, C3b and C4b, respectively. The precise functions of this receptor on lymphocytes are not known, but recent evidence supports some type of immunoregulatory role (4-7). CR2is a single glycosylated polypeptide chain with a molecular weight of 145 kd (8-10) which interacts preferentially with the terminal activation/processing fragments of C3, C3dg, and C3d. CR2is present on B lymphocytes and follicular dendritic cells in lymphoidorgans (9, 11). The precise functions of this receptor are not known either, but appear to be immunoregulatory, a point considered in this review. Beginning approximately 12 years ago with work by Jondal, Yefenof, and other workers in George Klein’s laboratory (12, 13), a number studies have suggested that Epstein-Barr virus (EBV), a humanherpesvirus, uses a complementreceptor to attach to, and to infect, B lymphocytes. The EBVreceptor and a complement receptor are coexpressed on multiple B cell lines and co-induced in negative cell lines; the same treatments induce cocapping and costripping from B cell membranes(12-16). These and other shared properties correlate with CR2rather than CR1 expression (17). Epstein-Barr virus, a lymphotropic humanherpes virus, is a significant humanpathogen. After primary infection, which may be inapparent or be manifest as infectious mononucleosis(18, 19), the virus becomeslatent a minor proportion of B lymphocytes and oropharyngeal cells; more than 90%of individuals in the USpopulation carry the virus. It is a candidate humancancer virus, as it is strongly linked to nasopharyngeal carcinoma and Burkitt’s lymphoma,two malignancies, and it is oncogenic in sub-

Annual Reviews

Annu. Rev. Immunol. 1988.6:85-113. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

IMMUNOBIOLOGY

OF CR2

87

humanprimates (20-22). In vitro it transforms humanB lymphocytes, generating immortalpolyclonal lymphoblastoidcell lines (14). Furthermore, EBVis associated with the X-linked lymphoproliferativesyndrome (Duncan’sdisease) (23), and it maycontribute to the development cell neoplasiain certain patients with AIDS (24). Finally, it is associated with several humanautoimmune diseases (25-27). In addition to its ability to transform normalhumanB lymphocytes, EBVis also a polyclonal B cell activator (28-31). In contrast to immune B cell activation, EBV-induced B cell activation does not require the presence of T cells or macrophages (29, 31), and it is not dependent prior immunityto the virus (32, 33). Immune T cells and T cell factors have the ability, however,to modulateB cell activation inducedby EBV and by the other ligands of CR2(5, 25, 34). This reviewcritically examines older as well as recent studies pertaining to the B cell membrane structure(s) to whichC3fragmentsand EBVbind, and it analyzesthe functional consequencesof these interactions. While we emphasizeour views that the CR2complementreceptor initiates EBV activation, infection, and transformation, and that interactions of this receptor with the physiologicC3ligands regulate B cell function, studies suggestingalternative interpretations are also considered.Ourgoal is to provide a conceptual frameworkboth for understanding the status of research concerning CR2and for the design of future experiments to elucidatethe biological functionsof this B cell surfaceglycoprotein. RELATIONSHIP AND CR2

BETWEEN THE EBV RECEPTOR

The EBVReceptor Selective binding of EBVto B lymphocyteshas been demonstratedby a number of techniques--including cyto-adherence with EBV-producing cells (35) and binding of radiolabeled EBV(36)--and by several direct andindirect fluorescenttechniques(I 3, 14, 37). TheEBV receptoris readily cappedon addition of an antibodyto EBV(12), indicating that the receptor is mobile in the plasma membrane.Binding of EBVto B lymphocytesis saturable and, on addition of unlabeled EBV,reversible (36, Figure 1). Thesefeatures, together with the specificity and moderatelyhigh affinity of the interaction, indicate that EBV bindingto B cells is receptormediated. Extracts of Raji cell membranes have been observedto block EBVbinding to fresh Raji cells. Antibodyto such extracts detected a prominent 150 kd band in Raji cell membraneextracts, as shownby Simmonset al (38).

Annual Reviews

88

COOPER,

MOORE & NEMEROW

Annu. Rev. Immunol. 1988.6:85-113. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

~"-" x 24

~o~

¯ 5x1(

~ 10

5x10’

7 1~I0

Peripheral Blood BCeils Figure 1 Binding of ~2~I-EBVto B lymphocytes. Increasing amounts of human B lymphocytes were reacted with a constant amount of ~2SI-EBV(O--O). Nonspeciflc binding, determined in the presence of a 100-fold excess of unlabeled EBV,is also shown(O--~).

The CR2 Receptor Althoughearlier shownto be distinct from other complementreceptors on the basis of functional criteria (39-41), the C3dbinding complement receptor, CR2,wasfirst characterizedas a discrete moleculeby Iida et al (9). Theseinvestigators isolated a 140 kd glycoproteinwith an isoelectric point of 8.2 from B cell membranesby affinity chromatography on Sepharose C3. This protein reacted with a B cell-specific monoclonal antibody, anti-B2, whichhad beenearlier developedby Nadleret al (42). Anti-B2partially inhibited C3drosetting, and this property was further amplified on addition of a polyclonal antibody to mouseimmunoglobulin. Shortly thereafter Weiset al (10) identified a secondB-cell-specificmonoclonal antibody, HB-5, whichwas reactive with a molecule of similar molecular weight; HB-5also inhibited C3d-dependentrosetting in the presence of a second antibody. Furthermore, protein A-containing particles preloaded with HB-5selectively adsorbeda 145-kd protein from labeled B lymphoblastoidcell membraneextracts. Neither anti-B2 nor HB-5interacted with the portion(s) of CR2responsible for binding C3d (9, 10~ 43). Other CR2reactive monoclonalantibodies termed OKB7 (36), AB1, AB2, AB3, and AB5(44), BL13(45), and RFB6(46) have subsequentlyidentified. Of these, only OKB7 effectively inhibits C3drosetting in the absenceof a secondantibody(36, 43, 47); polyclonalanti-CR2 antibodiesalso havethis property(48).

Annual Reviews

Annu. Rev. Immunol. 1988.6:85-113. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

IMMUNOBIOLOGY

OF CR2

89

CR2wasfirst isolated by Barel et al (48) by affinity chromatography Raji cell membraneextracts on C3 Sepharose, but it was erroneously characterized as a C3breceptor. At that time it wasnot knownthat Raji cells have CR2but lack CR1.CR2has also been purified from tonsil B lymphocytesby affinity chromatographyon immobilizedC3dg(49). and others have isolated the 145-kd CR2molecule in high yield and in functionally active form by immunoaffinity chromatographyon monoclonal antibodycolumns(50, 51). In contrast to these studies, Lambriset al (52) isolated a 72 kd glycoprotein (gp72) from the spent culture mediaof Raji cells by affinity chromatographyon C3dagarose. These workers found that rabbit antigp72 blocked C3d dependent rosetting to B lymphocytes and lymphoblastoid cells, but others, whohavealso isolated gp72and generated polyclonalantibody,wereunableto confirmsuch inhibition (53). Digestion of purified CR2and of Raji membraneextracts with trypsin and V-8 protease yielded multiple bands including somewith molecularweights in the 70-75 kd range, but these were not major fragments(43). Thus, the nature of gp72and the possibility that it is either a CR2fragmentor, alternatively, unrelated to CR2has not beenascertained. Relationship Between the EBV Receptor and CR2 Theevidencein favor of identity of these receptors is summarized below. Jondal, Yefenof, Einhorn, and other investigators workingwith George Klein (13-17) found the EBVand C3dreceptors coexpressedon multiple B cell lines. Theseinvestigators(12, 13, 55) also showed that the receptors capped together and independently of other B cell membranemarkers including surface Ig, HLA,and Fc receptors, and that both receptors were proteolytically stripped from the plasmamembrane by the sameenzymes. The receptors were also co-inducedin receptor negative cell lines from patients with undifferentiated lymphomas by treatment with theophylline (54). Their association was further suggestedby the observationthat blocked fusion of EBVwith B cell membranesas assayed by changes in fluorescence polarization (56). Furthermore,rosetting of C3d-coated particles with B cells wasimpairedby prior incubation of the cells with EBV (13, 57), and, in the reverse direction, sequential incubationof cells with C3followed by anti-C3 and anti-immunoglobulinreduced binding of virus (13). Althoughthe type of B cell complement receptor involved someof the earlier studies wasnot known,recent studies with monoclonal antibodies and well-defined reagents have clearly shownthat C3d-dependent rosetting is mediatedvia CR2and that Raji cells possess only CR2 (4, 9, 17,37, 58). Moredirect evidencefor identity of the EBVand C3dreceptors has come

Annual Reviews

Annu. Rev. Immunol. 1988.6:85-113. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

90

COOPER,

MOORE & NEMEROW

fromrecent studies of Fingerothet al (37). Theyfoundthat modulation CR2from the surface of B cells with the HB-5antibody, together with antimouseIg, blockedEBVbinding (37). In related studies, Tedderet (59) showedthat the same treatment impaired EBV-dependentB cell activation and Ig production. Protein Abearing S. aureusparticles preloaded with HB-5and incubated with detergent lysates of CR2positive cells were shownby Fingeroth et al (37) to bind radiolabeled EBV. have reported that the anti-CR2 monoclonal antibody OKB7 directly blocksbinding of C3dgmicrospheres,C3dbearing erythrocytes, and radiolabeled EBVto normal humanB lymphocytesand to Raji lymphoblastoid cells in the absenceof a secondantibody(36; Figure2). Consistentwith the

A 17.5.

7.5-

0.’250’.50.’751’.0 .88 z 72 I

I

56 E~ ,40 ~

0.2 0.6 1.0 1.4 1.8 Monoclonal Antibody Figure 2 Effect of monoclonal antibodies on 35S-labeled EBVbinding and infection. (A) Normaltonsillor B (left) or Raji (right) ceils (107) were reacted with various amounts OKB7(0--0) or anti-B2 (O --- O) antibody before the addition of 4000 cpm of labeled EBV.X Axes, monoclonal antibody (#g). (B) B cells (6 x 105) were reacted various amounts of OKB7(0) or anti-B2 (~,) antibody, washed, and then cultured for days in the presence of EBV.Infectivity was measured by the stimulation of DNAsynthesis ([3H]thymidine incorporation) and colony formation.

Annual Reviews

Annu. Rev. Immunol. 1988.6:85-113. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

IMMUNOBIOLOGY OF CR2

91

inhibition of binding, OKB7 (but not anti-B2) directly blocked infection of normal B cells (36; Figure 2). Polyclonal anti-CR2 has also been found to directly block EBV infection (53). Recently, we reported that purified CR2 binds to purified EBV and C3dg immobilized on nitrocellulose (50; Figure 3). Isolated CR2 did not interact with cytomegalovirus, another human herpesvirus, murine leukemia virus, or with ovalbumin, a protein with physicochemical properties similar to those of C3dg. Specificity was further shown by the ability of OKB7, but not another anti-CR2 monoclonal antibody, to block binding of purified CR2 to both ligands (50; Figure 3). In other studies, we also found that phosphatidylcholine liposomes into which purified CR2 had been incorporated possessed the ability to specifically bind purified EBV to cells expressing EBV antigens on their membrane and to C3d coated erythrocytes (60); this binding was also inhibited by OKB7 monoclonal

Figure 3 Inhibition of receptor binding by anti-receptor MAb. A total of 2 pg of purified C3dg (lanes 1 through 3, upper panel) or ovalbumin (lane 4, upper panel) or 10 ng of EBV (lanes 1 through 3, lower panel) or CMV (lane 4, lower panel) was coated onto nitrocellulose. The blots were then incubated with 40 ng of CR2 alone (lanes 1 and 4; upper and lower panels), with CR2 which had been preincubated with 6 pg of OKB7 MAb (lane 2, upper and lower panels), or with AB-I MAb (lane 3, upper and lower panels). Reactivity was detected by the biotin-avidin system.

Annual Reviews

Annu. Rev. Immunol. 1988.6:85-113. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

92

COOPER,

MOORE & NEMEROW

antibody. Finally, study of somatic cell hybrids showed that the genes coding for CR2and the EBVreceptor cosegregate (17). The ability of the OKB7monoclonal antibody to bind to a single 145kd B cell membraneprotein, together with the ability of the purified 145kd B lymphocyte membrane protein to bind specifically to both EBV and C3dg unequivocally demonstrates that the 145-kd molecule is a dual receptor for both ligands. Since OKB7directly blocks EBVbinding to, and infection of, lymphocytes, CR2serves as a B cell receptor mediating both EBVpenetration into the cells and EBVmediated transformation. These data do not, however, unequivocally rule out the existence of either an additional EBVreceptor or another C3dg receptor on the same cells, although it is clear that such receptor(s), if present, can maximallymediate the binding of 10%of the added EBV(36; Figure 2). There are, however, other studies that suggest that the CR2complement receptor and the receptor mediating EBVbinding and transformation are not identical. Several of the results appear to be explainable on the basis of subsequently derived knowledge. One such study (57) used the above mentioned 72-kd B cell membrane as CR2. Gp72 neither inhibits EBV binding to, nor infection of, B cells (57), and antibodyto it does not inhibit EBVbinding (53). It clearly is not the CR2molecule that has been well characterized by manyinvestigators. Thus, the finding that human/mouse somatic cell hybrid clones (made from gp72/EBVreceptor positive human cells and gp72/EBVreceptor negative murine myelomacells as parents) express either gp72 or EBVreceptor function but rarely both properties (57) is not relevant for assessing the identity of CR2and the EBVreceptor. Wells et al (61) concluded that the EBVreceptor and CR2were different structures, on the basis of differential inhibition of virus and C3dbinding to various lymphoblastoid cells by various antisera to complementreceptors. These data were not corrected for the different numbers of EBVreceptors on the various cell types. Other interpretations of this study are also possible, notably that C3d and EBVbinding are properties of different epitopes on the same molecule. Several studies have noted the ability of phagocytic cells to bind EBV (62, 63). Such cells cannot be infected with EBV.The C3d binding structure on neutrophils and monocytesis a structurally different molecule (4, 63, 64). Certain T cell lines, null cells, and undifferentiated T cells bind EBV and express functional C3d receptors (4, 65, 66). In the case of the T cell line Molt-4, this has been found to reflect the presence of antigenically detectable CR2on the cells (8, 37). The nature of the C3d/EBVbinding structure on certain other cell types, which cannot be infected with EBV, remains to be determined. A numberof studies are presently unexplainable on the basis of the 145-

Annual Reviews

Annu. Rev. Immunol. 1988.6:85-113. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

IMMUNOBIOLOGY OF CR2

93

kd CR2moleculeas the only EBVand C3dreceptor on B cells. The Ramos cell line, whichdoes not contain the EBVgenome(67), bears functional C3dreceptors and EBVreceptors (12, 67) but does not react with antiB2(68). EBVinfection inducedB2expression on these cells (69). These data are not presently explainableon the basis of CR2as a single C3dand EBVreceptor, but it should be kept in mind that anti-B2 only weakly stains mostB cell lines andlow’levelsof reactivity mighthavebeenmissed. Differential expressionof different CR2epitopesis also possible. Somestudies with pre-Bcells also do not readily support the existence of a single receptor for C3dand EBV,but results of several studies in this area are uninterpretable. For example,pre-Bcells from fetal bonemarrow andliver havebeenreported by Tedderet al (8) andby Bofill et al (70) lack the HB-5and RFB6CR2antigens, respectively. However,Hansson et al (71) were able to infect fetal bone marrowcells with EBV,and the derivedcell lines containedlow levels of the B2antigen and rosetted with C3dbearing erythrocytes. Similarly, Katamineet al (72) also derivedsome pre-B cell lines from fetal liver of the sameage by EBVinfection which possessedfunctional C3dreceptors. Hibi et al (73) also wereable to infect pre-B cells from fetal bone marrowwith EBV.B cell lines with pre-B phenotypewhich were generated from bone marrowof patients with Xlinked agammaglobulinemia by EBVinfection by Fu et al (74) contained functionalC3dreceptors. Clearly, the ability to infect pre-Bcells with EBV is in apparent contradiction with the lack of detectable CR2antigen on these cells. It is importantto keepin mind,however,that EBV infectibility and the fluorescent techniquesused to detect CR2antigens are unrelated techniqueswith very different sensitivities. TheEBV-infected cell lines may have represented the progenyof only a few cells or a subpopulationof cells bearing EBVreceptors and possibly CR2whichescapeddetection by immunofluorescence. Alternatively, both Tedderet al (8) and Bofill et (70) havereported the presenceof cells in certain fetal tissues (spleen, lymphnode, and tonsil) of the sameage that were CR2antigen positive; a fewsuch cells, if also present in bonemarrowor liver, could represent the cells that wereinfected by EBV.In addition, since EBVinfection is a single hit process (75, 76), a single EBVvirion and therefore only a few receptors on a pre-Bcell are neededfor infection. Suchlevels could have escaped detection by immunofluorescence.Still other explanations are possible, e.g., (a) the receptors maydiffer on pre-Bcells; (b) pre-Bcells maybe infected in a different mannerby EBV,or (c) there may differential expression of EBVreceptor and CR2epitopes on fetal pre-B cells. Also unexplainableat present is the absenceof EBVreceptors and EBV noninfectibility of B cells from patients with the common, nonsecretory

Annual Reviews 94

COOPER,

MOORE & NEMEROW

Annu. Rev. Immunol. 1988.6:85-113. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

form of agammaglobulinemia coupled with the presence of C3dreceptors on their B cells, as assessedby C3d-dependent rosetting (77). In the reverse direction, a single cell line established by EBVinfection of normalperipheral bloodB cells failed to express CR2whetherassayedby reactivity with anti-B2, HB-5,or by C3d-dependent rosetting; but it did express low levels of EBVreceptors (78). Similarly, cell sorting studies showedthat significant proportion of the cells susceptible to transformationby EBV werepresent in the anti-B2negativecell population(79). CELL AND TISSUE DISTRIBUTION EBV RECEPTOR AND CR2

OF THE

EBVwas early demonstratedto selectively infect normal humanB lymphocytesin vitro, and this led to Ig productionandindefinite proliferation (29, 35). EBVprimarily infects mature, high density, resting, GoB lymphocytesbearing complement receptors (80-82). Therelationship between complement and EBVreceptors has been considered in the section above. EBVtransformedB cell lines derived from normalB cells are polyclonal and vary in size, morphology,and expressionof surface markers; all have diploid karyotype(68, 80, 83). Their surface markerpattern resemblesthat of mitogen-activatednormalB cells, and such cell lines often expressIgM and IgDand secrete large amountsof Ig (80, 84). Althoughtransformation of single cells follows single hit kinetics, only a minor proportion of complementreceptor positive lymphocytesbecometransformed (28, 32, 74, 75, 76). EBVinfection is capableof inducingonly limited maturation, suchas inductionof Ig synthesis; it does not lead to Ig generearrangement or Ig class switch, although more pronouncedmaturation mayoccur on occasion (69). Thus, individual cells transformedby EBVsecrete the characteristic of the stage of development wheninfection occurred(25, 72, 75, 85). Burkitt lymphoma-derived cell lines, whetheror not EBVinfected, are monoclonaland never have a diploid karyotype; they also exhibit chromosomaltranslocations involving Ig loci and the c-myc oncogene (80); mostare surface IgDnegative andpoor Ig secretors (68). Mostbut not all bloodand tissue B cells, B lymphoblastoidcell lines, and B cells frompatients with B cell leukemiasand lymphomas react with anti-CR2monoclonalantibodies (8, 42), while bloodand tissue monocytes, NKcells, granulocytes,and normaland activated T cells as well as T cell lines (except Molt-4 and cells frompatients with certain T cell malignancies) do not. Monocytesand neutrophils also contain a C3d-binding molecule(62, 63), but the structure of this moleculeis entirely different from CR2and has been termed CR4(4, 63, 64). A recent report (86)

Annual Reviews

Annu. Rev. Immunol. 1988.6:85-113. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

IMMUNOBIOLOGY

OF CR2

95

suggests that platelets might possess CR2,but the possibility of sufficient B cell contamination to explain these results was not rigorously excluded; others have failed to find CR2on platelets (87). In lymphoidtissues significant differences in the reactivity of various monoclonal anti-CR2 antibodies have been observed. Anti-B2 reacts weaklywith B cells in the peripheral area of primary follicles and in the mantle zone of secondary follicles, while reacting intensely with cells in the germinal centers (88). Other antibodies with likely similar or identical specificity, BL13and RFB6,have a similar pattern of reactivity (45, 46). Anti-B2reacts weaklywith peripheral blood B cells (42, 88, 89). In contrast, HB-5reacts strongly and homogeneously with both germinal center and mantle zone cells in the secondaryfollicle (45); it also reacts strongly with peripheral blood B cells (8). Somecells in T dependent areas are also stained with HB-5but not with BL13(45). In addition, anti-B2, BL13, and RFB6have been found to stain follicular dendritic cells in germinal centers (11, 45, 46, 88). The EBVgenomeis found in epithelial cells taken from patients with nasoepithelial carcinoma (90, 91), although numerous attempts to show EBVreceptors on nasoepithelial cells or to infect the cells with laboratory EBVadapted strains have been unsuccessful (92, 93). Studies in which the membranebarrier was bypassed by implantation of receptor or by other techniques have shown that such cells can be infected with EBV(93). Sixbeyet al (94) wereable to infect primary epithelial explant cultures with EBVpatient isolates but not with laboratory adapted EBVstrains as manifested by induction of EBVDNAand EBVVCAantigens in the cells. Recent studies with two anti-CR2 monoclonal antibodies, HB-5and antiB2, have shown that both antibodies react with humanpharyngeal epithelium in a cell differentiation-dependent manner (95). These findings point to the clear possibility that EBVmaydirectly enter and infect human epithelial cells at particular stages of differentiation in vivo. Further study in this area is needed. CR2 EXPRESSION DEVELOPMENT, DIFFERENTIATION

DURING B CELL ACTIVATION AND

Despite the relatively restricted numberof B cell functions, whichlargely center on antigen presentation, antibody production, and induction of B memorycells, B cells are remarkably heterogeneous in terms of the number and diversity of molecules that can be detected on their membranes.B cell development from stem cells to pre-B cells, early B cells, and mature B

Annual Reviews

96 Stem Cell

COOPER, Pre-B Cells

MOORE & NEMEROW Immature¯ B Cell

Mature B Cell

Annu. Rev. Immunol. 1988.6:85-113. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

B Cell Development (Antigen-independent)

Activated B Cells

Plasma Cell

B Cell Differentation ~ ,,--=(Antigen or polyclonal~ activator dependent)

Figure 4 B cell maturation/activation/differentiation sequence. Surface structures are shown./z denotes IgM heavy chain; CR1 and CR2, the complement receptors; Ia, MHC class II antigens; FcR the Fc receptor; Grf, receptors for growth factors; TID, a 35 kd glycoprotein and Y, complete immunoglobulin molecules.

cells, is often considered to be a linear progression (96-99) and is presented as such in Figure 4. The expression of many B cell membranemarkers changes dramatically during B cell maturation, activation, and differentiation. CR2 Expression

Duriny

B Cell

Development

The ability to bind EBV, to rosette with C3d-bearing particles and to react with monoclonal antibodies to CR2have been described as B cell development/differentiation markers restricted to a limited portion of the B cell development sequence (8, 42, 46, 89). However, as noted earlier, various investigators have reported differential expression of these three properties by fetal B cells (8, 70-74). Variable expression of CR2antigens by pre-B cells from various tissues has also been observed (8, 70). Also, adult pre-B cells have been found by Hibi et al (73) to express the CR2 antigens detectable with anti-B2 and HB-5, and to be infectible with EBV, while Campanaet al (46) reported such cells to be negative for CR2 assessed with the RFB6anti-CR2 monoclonal antibody. Clarification of the ontogeny of CR2requires further study. Tosato et al (100) have derived a cell line from normalperipheral B cells by EBVinfection that lacks intracytoplasmic and membraneIg and thus resembles an immature B cell. However, since the cell line contained rearranged immunoglobulin genes, it is more likely derived from a more mature B cell that has undergone ineffective Ig rearrangement or contains a defect in transcription of the Ig genes. Its lack of detectable CR2as assessed with anti-B2 or HB-5is unexplained but could reflect the loss in CR2that occurs on proliferation of activated B cells (see below).

Annual Reviews IMMUNOBIOLOGY

OF CR2

97

Annu. Rev. Immunol. 1988.6:85-113. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Alterations in CR2 Expression During B Cell Activation and Differentiation Changesin B cell expression of CR2during B cell activation were first reported by Stashenkoet al (89). Theseworkers assessed reactivity humanperipheral blood B cells with anti-B2 after activation with pokeweedmitogen (PWM).CR2expression transiently increased on day 3 and then disappeared by day 4-5. Activated humanspleen and tonsil B cells lost CR2with the samekinetics but did not exhibit the transient increase observedwith the peripheral B cells. CR2loss wastemporally associated with loss of surface IgD, development of intracytoplasmicIgM, and expression of a plasmacell marker(Figure 4). TheB1 antigen also disappeared 6-7 days after activation with PWM, coincident with the appearance of intracytoplasmic and membrane IgG (C6), Similar loss anti-B2reactivity by splenic B cells followingactivation with anti-Ig has beenshownby Boydet al (101) and by others (8, 88, 89). Markedreduction in B2antigen expression has also been observed18 hours after EBV-induced B cell activation (102), likely reflecting the same process although internalization of the receptor together with EBVwas not excludedin_ these studies. MatureB cells as well as spontaneously activated large B cells bind and internalize EBVand mayproduce Ig althoughthey do not alwaystransform(32, 73, 80, 81). Further study has shownthat while B cells in the resting state (Go)or newlyactivated with mitogen or anti-# (G1) can be transformed with EBV,cells that have entered the S phaseof the cell cycle do not (82, P. Casali, personal communication).Thesestudies indicate that CR2is lost with activation and entry into the cell cycle and proliferation. It has beendocumented with anti-B2 (88, 89) and HB-5(8) that plasmacells lack CR2(Figure

PHYSIOCHEMICAL ANALYSIS STRUCTURE AND ANTIGENIC

OF CR2 EXPRESSION

Biosyntheticapproaches(103) as well as enzymaticstudies (43) haveshown that CR2is derived from a 111-l15-kd nonglycosylated precursor molecule. The fully glycosylated 145-kdCR2glycoprotein contains complex N-linked but not O-linked oligosaccharides (103). Nonglycosylated CR2, high mannose containing CR2, and mature CR2have all been observed to bind to Sepharose-conjugatedC3, indicating that carbohydrate residues are not necessary for CR2function. The presence of oligosaccharides was also found to be unnecessaryfor plasma membrane expression of CR2(103). However,the half-life of nonglycosylatedCR2

Annual Reviews

Annu. Rev. Immunol. 1988.6:85-113. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

98

COOPER,

MOORE & NEMEROW

on the plasma membrane was greatly reduced compared to the mature molecule. CR2has been assigned to the CD21antigen cluster of B cell differentiation.(104). Four monoclonal antibodies to CR2(OKBT,HB-5, antiB2, and AB-1) have been found to recognize distinct and nonoverlapping antigenic epitopes on the CR2molecule (43); anti-B2 and HB-5had been earlier found to be nonoverlapping (8). Another anti-CR2 antibody, BL13, has been found to recognize an epitope closely associated with or identical to the B2 epitope, and nonoverlapping with HB-5(45). The RFB6 anti-CR2 monoclonal antibody has a pattern of reactivity with B cells similar to that of anti-B2 (46, 70). Several of these m0noclonalantibodies have been found to have different functional properties. Thus, OKBT,but neither HB-5(36, 37), anti-B2 (36), or AB-1(34), directly blocked EBVbinding to and infection lymphocytes in the absence of a second anti-Ig antibody (Figure 2). addition, OKB7(37) but not HB-5 (10, 34) also directly blocked function; anti-B2 (9, 34), BL-13(45), and AB-1(34, 44) had limited ability to do so. OKB7(34) and AB-1 (34, 44) have also been observed to mitogenic for B lymphocytes in the presence of T cells; anti-B2 had some activity in this regard while HB-5was completely inactive (8, 34). These various data indicate that the 145-kd B cell EBV/C3dreceptor possesses discretely structural domainswith distinct functional correlates. OKBT, HB-5, and AB-1 also fully immunoprecipitate CR2 from lymphoblastoid B cell membraneextracts, while anti-B2 precipitates only a portion of the membraneCR2(43). Also anti-B2 (42, 88, 89), BL-13(45), and RFB6 react only with some B cells in lymph nodes and weakly with peripheral blood B cells, while HB-5(45) reacts strongly with all B cells in tissues and with peripheral blood B cells as noted earlier. These latter observations suggest that differential expression of CR2epitopes mayoccur on someB or subsets of B cells. MOLECULAR THE EBV/C3d

CLONING OF RECEPTOR

THE

cDNA

ENCODING

Weis et al (105) isolated a partial cDNACR2clone from a tonsil B cell ¯ library cloned in 2gt 11. Althoughrelatively little CR2nucleotide or amino acid sequence was presented in the report, several important observations were made. The mRNA-encodingCR2was shown to be approximately 5.0 kb by Northern blot analysis. CR1and CR2were found to be structurally related to the DNAlevel, since a CR1cDNAclone cross-hybridized with

Annual Reviews

Annu. Rev. Immunol. 1988.6:85-113. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

IMMUNOBIOLOGY

OF CR2

99

CR2eDNA at low stringency (105). At the protein level, there is sequence homology, and both show a 60-75 amino acid consensus repeat motif containing6-10 conservedresidues. This structural feature is foundin six other C3binding proteins (106), as well as in several unrelated proteins includingthe interleukin-2receptor (106, 107). Klicksteinet al (108) proposeda CR1modelin whichthe 60-75 aminoacid repeating elements are arranged linearly to form a highly elongatedmembrane protein extending 500 A~fromthe cell surface. This structural modelhas not yet been verified. The genes encodingboth CR1and CR2are located on the long arm of humanchromosome 1 at band 1Q32(109) suggesting that these receptors mayhave arisen from a common ancestral gene through duplication and that there maybe a complement receptor locus on chromosome 1. The other non C3-bindingproteins sharing the 60-75 aminoacid consensus repeat are on other chromosomes (109). Wehave also employed2gtl 1 for the molecular cloning of eDNA encoding the EBV/C3dg receptor (M. D. Moore, N. R. Cooper, G. R. Nemerow,manuscript submitted). ComplementaryDNAwas synthesized from polyadenylatedmRNA from Raji B lymphoblastoidcells. The library wasscreened with a uniquesequence39meroligonucleotide probereported by Weis et al (105) to correspondto a region in the 5’ portion of CR2 cDNA.Withthis probe we isolated two clones designated 2A11and 2F11 whichhybridizedto the probeon three successiveroundsof screening. As shownin Figure 5, both of these clones containedEco Rl-derivedinserts of approximately1.2 kb. As shownin Figure 5, the isolated ,~A11insert hybridized to a 4.7-kb RNAfrom Raji B cells on Northernblots but not from humanHSB-2T cells. Theseproperties are consistent with the size and distribution previously reported for CR2mRNA. Using the 1.2-kb insert, the library was rescreened and two morecDNA clones designated 2E41 and 2Lll were isolated. As shownin Figure 6, the )~E41 clone contained Eco R1derived inserts of approximately1.8, 1.6, and 0.8 kb (4.2 kb total) whilethe 2L11clonecontaineda single insert of 1.2 kb. The 1.8 kb Eco R1fragmentof clone E41and the 1.2 fragmentof clone L11 hybridizedto the hA11insert on Southernblot analysis (Figure 6). Escherichiacoli wereinfected with purified ~,E41phageand inducedto synthesize protein. Plaques containing recombinantprotein were replicated onto nitrocellulose filters and screenedwith either rabbit anti-CR2 or normal rabbit serum. Immune (but not nonimmune serum) reacted with 2E41plaques; other control plaques generatedby irrelevant clones failed to react. Approximately90%of the 4.2-kb 2E41.CR2clone has been sequenced on both strands. A summaryof the restriction mappingand sequence

Annual Reviews

Annu. Rev. Immunol. 1988.6:85-113. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

100

COOPER, MOORE & NEMEROW

Figure 5 Restriction enzyme and Northern blot analysis of ICR2 cDNA clones. IAl1 and IF1 1 DNA was digested with Eco R1, electrophoresed on a 1% agarose gel and stained with ethidium bromide (A). The isolated 1.2-kb insert of I A l was nick translated with "P and used to probe total RNA (20 pg) or poly A + RNA (2 pg) from CR2 positive Raji cells or CR2 negative HSB-2 cells (B).

analysis of the CR2 clones is shown in Figure 7. A major feature of the CR2 sequence is that the entire extracellular domain excluding only the putative signal peptide is comprised of tandem 60-75 amino acid repeat motifs (Figure 7) which share the above noted homology with a number of C3-binding and other proteins. The E41.CR2 cDNA encoding CR2 contains 16 such repeating elements.

Annual Reviews

Annu. Rev. Immunol. 1988.6:85-113. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

IMMUNOBIOLOGY OF CR2

101

Figure 6 Restriction enzyme and Southern blot analysis of 1CR2 cDNA clones. LE41 and LLl I cDNA clones were digested with Eco R1 and analyzed on a 1.5% agarose gel (left panel). '%-labeled LA11 insert was used to probe a Southern blot of the same gel (right panel). 1-kb ladder and 123-bp ladder markers were run in lanes 1 and 2, respectively.

BIOLOGIC FUNCTIONS OF CR2 B cells serve as effective antigen presenting cells. In addition the B lymphocyte and its end-stage differentiation product, the plasma cell, are the sole producers of antibody molecules. Finally, the B cell also gives rise to the memory cell which is in part responsible for the rapid, heightened appearance of specific antibody on secondary encounter with antigen.

Annual Reviews 102

COOPER, 1~

MOORE & NEMEROW 2k~

I

I

3kb

~

4kb

4.7kb

l 1~

CR2 mRNA

3 Probe AAII, IFII, ALl1

l.Skb

1.6kb

0.Skb i

Annu. Rev. Immunol. 1988.6:85-113. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

~ ,, ,

NH2

COOH

~I f~, J I I I I I I I I I [ I I I I K-~ Signal Figure 7

~E41

CR2 Protein

Transmembran’e Restriction

map of 2CR2 clones

and the deduced CR2 protein.

Such functions of B cells are generally perceived to be dependent on various types of interactions betweenB cells, T cells, and accessory cells together with soluble growthand regulatory factors secreted by these cells, although the molecular mechanismsinvolved have not yet been elucidated. The CR2 receptor on B cells is undoubtedly involved in one or more of the abovelisted B cell functions. It is appropriate at this point to review the studies that documentvarious functional properties of CR2and CR2ligands. CR2is present on B cells only during a restricted portion of the B cell maturation sequence and disappears uponentry of B cells into proliferation; it also disappears with terminal differentiation into plasmacells (8, 42, 46, 89). Thus, it clearly cannot play a role either in the earliest or latest stages of B ¢¢11differentiation. Someinsights into the functions of this receptor can be gleaned from studies with EBV. EBValone does not induce Ig heavy chain gene rearrangement and isotype switching; it also does not induce terminal differentiation since EBVtransformed Ig secreting cells are morphologically and phenotypically different from plasma cells (25, 72, 75, 85). EBVdoes, however, induce some differentiation since EBVinfected cells acquire the ability to secrete significant amountsof Ig (25, 28, 75, 76, 110). Induction of indefinite B cell proliferation is independent of the transforming activity of EBVbecause only a minor proportion of B cells induced by EBVto proliferate go on to form immortal cell lines (32, 73, 110). Furthermore, the nontransforming P3HR1EBVstrain induces

Annual Reviews

Annu. Rev. Immunol. 1988.6:85-113. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

IMMUNOBIOLOGY OF CR2

103

proliferation without immortalization (25, 111) as does the UV-irradiated transforming MCUV5 EBVstrain (112). These proliferative effects defective EBVrequire T cell factors (25, 111, 112). Other CR2ligands also have the ability to activate or modulate B cells. Thus, Sepharose-bound humanC3d stimulates preactivated immuneB cells to proliferate (5, 113) and soluble C3dinhibits proliferation in the presence of appropriate monocyte-derived growth factors (5). Certain monoclonal antibodies to CR2 and polyclonal anti-CR2 induce B cell proliferation and Ig secretion (34, 44, 114), effects that require T cells or T cell factors. The OKB7 monoclonal antibody has also been found to modestly enhance the generation of Ig secreting cells in a PWM driven system (115). However,these systems are complex, and manyaspects are not understood. Thus, the role is unclear of monocytefactors, indicated as necessary for stimulation of murine B cells by cross-linked C3d(5, 113), and of T cells or T cell factors found be required for proliferation of B cells by nontransforming EBV(25, 11 l, 112) and monoclonal and polyclonal anti-CR2 antibodies (34, 44, 114). This is particularly true since transforming EBValone is a highly efficient T cell-independent B cell activator (29, 31). Also it is not understoodthat murine B cells apparently require preactivation in order to be stimulated by Sepharose-bound C3d (5, 113), but this is not required for human cell activation by transforming EBV(29, 31), nontransforming EBV(25, 111, 112), and antibodies (34, 44, 114). There are other complexities: contrast to their behavior with preactivated murine B cells, Sepharosebound C3d and cross-linked C3d do not stimulate human B cells to proliferate either directly or in the presence of T cell factors; nor do they modulate B cell activation produced by other factors (59; J. F. Bohnsack, G. R. Nemerow,N. R. Cooper, unpublished observations). Also some but not other antigenic epitopes on CR2stimulate B cell activation; thus, OKB7and AB-1activate while anti-B2 and HB-5 do not (8, 34, 44, 59). Furthermore, induction of B cell activation by OKB7is variable since earlier but not later batches of OKB7 have been found stimulatory (34; J. F. Bohnsack, G. R. Nemerow,N. R. Cooper, unpublished observations). Despite these uncertainties, collectively these various studies indicate that under certain circumstances CR2ligands may either produce or modulate B cell activation. Also pointing to the involvement of CR2in a B cell activation pathway is the finding that CR2becomes phosphorylated on activation of human B cells with phorbol ester (116), anti-#, or S. aureus (117). Whetherthe natural ligands, EBVand C3d, or other ligands have the ability to induce phosphorylation is not known. Since CR2does not autophosphorylate (116, 117) and the cytoplasmic tail of CR2is too short itself to serve

Annual Reviews

Annu. Rev. Immunol. 1988.6:85-113. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

104

COOPER,

MOORE & NEMEROW

a protein kinase (see above), another protein kinase must be responsible. The CR2receptor is mobile in the plasma membraneof B lymphocytes, as is indicated by the ability to cap EBVon the B cell with anti-EBV(12). This is also true of CR2on addition of HB-5followed by anti-Ig (10). have shownthat CR2mediates internalization of EBVinto normal B cells and B lymphoblastoid cells (118, Figure 8). With normal B cells, this process proceeds via an endocytic pathway and involves the participation of multiple elements of the cytoskeleton (118, 119; G. R. Nemerow,N. Cooper, unpublished observations). In addition, EBVinternalization is calmodulin dependent, indicating also a calcium requirement (119). Calcium is required also for multiple aspects of transmembranesignalling in B lymphocytes (reviewed in 120). These findings lend further support the involvementof this receptor in B cell activation. The natural ligands for CR2are C3 activation/processing fragments. Manystudies over the past 15 years have suggested that complement, and particularly C3, regulates certain immunereactions. There are repeated findings that C3 fragments modulate numerous B and T cell responses in vitro (121-123), and it was suggested early that C3 might mediate T-B cell collaboration since responses to certain T dependent antigens were impaired in the absence of C3 both in vivo (124-126) and in vitro (126). This dependence is not absolute, however, since C3 not essential for many T dependent responses (127). Its role in T dependent antibody responses is thus unclear. Early reports that C3 might provide an essential second signal (in addition to antigen) for T cell-independent B cell triggering (126) were disproved by manystudies showing that antigen and C3 binding to B cells are not sufficient for triggering (128, 129). Early as well as subsequent studies have indicated that C3 facilitates antigen localization in lymphoidfollicles since antigen, antibody, and C3 colocalize there normally (127) but not in C3 depleted animals (130). memory cells also are found in germinal centers (131, 132), and this process is C3 dependent (127, 130, 131). One characteristic feature of germinal centers, which develop in peripheral lymphoid tissues after immunization, is the presence of a unique cell type, the follicular dendritic cell (FDC). These cells possess complementreceptors, including CR2(l l, 45, 46, 88) and retain undegraded antigen-antibody complexes, likely as antigenantibody-complement complexes, on their surface for a long time (133). CR2are also on B cells in the germinal centers and in the mantle zones in secondary lymphoidfollicles (11, 46, 88). These observations suggest that C3 fragments attached to complement receptors on B cells and on FDC may participate in the generation of B memorycells in lymph node germinal centers (132, 134).

Annu. Rev. Immunol. 1988.6:85-113. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Annual Reviews IMMUNOBIOLOGY OF CR2

105

Annual Reviews

106

COOPER,

MOORE & NEMEROW

Annu. Rev. Immunol. 1988.6:85-113. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

CONCLUSIONS Muchremains to be learned about the biologic functions of CR2. It is important to keep in mind that the natural ligands of this receptor are the terminal processing fragments of C3 activation, C3dg or C3d. These fragments remain covalently bound to complement activators. Most immunecomplexes activate the complementsystem and therefore bear C3 activation/processing fragments. It is not generally appreciated that many and probably most pathogens and substances with pathobiological potential directly activate complement,in the absence of antibody, although antibody can augment the activation (1, 2). Thus, immunecomplexes well as pathogenic agents are likely coated with complementfragments, including C3dg, in vivo and thus have the potential to bind to CR2on FDCand B lymphocytes. It is tempting to speculate, as have Klaus & Humphrey(132, 134), that binding of immunecomplexes and complement activating pathogens to FDCin lymphoid tissues contributes to the generation of B cell memory.This is a particularly appealing hypothesis in view of the C3 dependence of B memorycell induction, the location of FDCin germinal centers in lymphoid organs, and the ability of FDCto retain surface antigen for prolonged periods of time. Surface Ig antigen receptors on B cells can provide the specificity that complementlacks. B cells with the appropriate specificity could potentially react with antigens in the immunecomplex or pathogens which have reacted, via C3dg with CR2on the FDC, as also postulated by Klaus & Humphrey(132, 134). Separate from these considerations, CR2ligands in the presence of T cells or T cell factors either modulate B cell activation or induce limited proliferation and Ig secretion, as noted earlier. Consistent with these functions are the findings that B cell activation is accompaniedby CR2 phosphorylation and that CR2is a participant in an endocytic pathway which is calcium and calmodulin dependent. Phosphorylation plays a role (in a manneras yet incompletely understood) in signal transduction and the regulation of numerousintracellular processes within cells, particularly those involving growth and other differentiated cellular functions; calcium and potentially calmodulin-dependent processes also function in B cell activation. These various findings point to a role for CR2in cross-membrane transmission of B cell surface signals. Furthermore, the ability of CR2ligands, with T cell help, either to activate, or modulate the stimulation of B cells implies that CR2fulfills similar functions in B cell activation as it normally occurs during antigenic triggering of immune responses. NormalB cell activation, initiated by antigen binding to B cell antigen receptors, is dependent upon and regulated by cell-cell contacts and the actions of multiple factors emanating from T cells and accessory

Annual Reviews

Annu. Rev. Immunol. 1988.6:85-113. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

IMMUNOBIOLOGY OF CR2

107

cells. Immunecomplexes and other complementactivating pathogens with pathobiological potential have the potential to interact simultaneously with antigen receptors and also complementreceptors not only on B cells but also on T cells and accessory cells. Ampleopportunities exist for complementreceptor-mediated regulation not only of B but also of T cell activation. Manylaboratories are using modern biochemical and genetic techniques to explore the molecular features and functional consequences of cognitive interactions between cells and regulatory cytokines of the immunesystem. These studies will undoubtedly also provide insights into the immunoregulatory functions of C3 and of CR2. ACKNOWLEDGMENTS

The authors wish to thank Bonnie Weier for her assistance in preparation of the manuscript, Publication No. 4953-IMM.This research was funded by NIH Public Health Service grants: AI17354, AI25016, CA14692, CA36204, PEWScholars Award for Biomedical Science.

Literature Cited 1. Cooper, N. R. 1987. The complement system. In Basic and Clinical Immunology, ed. D. P. Stites, J. D. Stobo, J. V. Wells, pp. 114-27. New York: Lange. 6th ed. 2. Miiller-Eberhard, H. J., Miescher, P. A., eds. 1985. Complement. NewYork: Springer-Verlag. 474 pp. 3. Fearon, D. T., Wong, W. W. 1983. Complementligand-receptor interactions that mediate biological responses. Ann. Rev. ImmunoL 1:243-71 4. Ross, G. D., Medof, M. E. 1985. Membrane complement receptors specific for bound fragments of C3. Adv. ImmunoL 37:217-67 5. Melchers, F., Erdei, A., Schulz, T., Dierich, M. P. 1985. Growthcontrol of activated, synchronized murine B cells by the C3d fragment of human complement. Nature 317:264-68 6. Daha, M. R., Bloem, A. C., Ballieux, R. E. 1984. Immunoglobulin production by human peripheral lymphocytes induced by anti-C3 receptor antibodies. J. Immunol. 132:1197-1201 7. Weiss, L., Delfraissy, J. F., Vazquez, A., Wallon, C., Galanaud, P., Kazatchkine, M. D. 1987. Monoclonal antibodies to the humanC3b/C4b receptor (CR1) enhance specific B cell differentiation. J. lmmunoL138:2988-93

8. Tedder, T. F., Clement, L. T., Cooper, M. D. 1984. Expression of C3d receptors during human B cell differentiation: lmmunofluorescence analysis with the HB-5 monoclonal antibody, J. Immunol. 133:678-83 9. Iida, K., Nadler, L., Nussenzweig, V. 1983. Identification of the membrane receptor for the complement fragment C3d by means of a monoclonal antibody. J. Exp. Med. 158:1021-33 10. Weis, J. J., Tedder, T. F., Fearon, D. T. r 1984. Identification of a 145,000 M membraneprotein as the C3d receptor (CR2) of humanB lymphocytes. Proc. Natl. Acad. Sci. USA81:881-85 l 1. Reynes, M., Aubert, J. P., Cohen,J. H. M., Audouin,J., Tricottet, V., Diebold, J., Kazatchkine, M. D. 1985. Human follicular dendritic cells express CR1, CR2, and CR3 complement receptor antigens. J. Immunol. 135:2687-94 12. Yefenof, E., Klein, G., Jondal, M., Oldstone, M. B. A. 1976. Surface markers on humanB- and T-lymphocytes. IX, Two-color immunofluorescence studies on the association between EBV receptors and complementreceptors on the surface 17:6923-7000 of lymphoidcell lines, lnt. J. Cancer 13. Jondal, M., Klein, G., Oldstone, M. B. A., Bokish, V., Yefenof, E. 1976.

Annual Reviews

Annu. Rev. Immunol. 1988.6:85-113. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

108

COOPER, MOORE& NEMEROW

Surface markers on human B and T lymphocytes. Scand. J. lmmunol.5: 401-10 14. Einhorn,L., Steinitz, M., Yefenof,E., Ernberg,I., Bakaes,T., Klein, G. 1978. Epstein-Barr virus (EBV)receptors, complement receptors, and EBVinfectibility of different lymphocyte fractions of humanperipheral blood. Cell. Immunol.35:43-58 15. Klein, G., Yefenof,E., Falk, K., Westman, A. 1978. Relationship between Epstein-Barr virus (EBV)-production and the loss of the EBVreceptor/ complement receptor complexin a series of sublines derived from the same original Burkitt’s lymphoma.Int. J. Cancer 21:552q50 16. Yefenof, E., Klein, G., Kvaruung,K. 1977. Relationships betweencomplement activation, complementbinding and EBVabsorption by human hematopoietiecell lines. Cell. Immunol. 31: 225-33 17. Jonsson,V., Wells,A., Klein, G. 1982. Receptors for the complement C3d componentand the Epstein-Barr virus are quantitatively coexpressedon a seres of B cell lines and their derived somaticcell hybrids.Cell. Immunol.72: 263-76 18. Henle, G., Henle, W., Diehl, V. 1968. Relation of Burkitt’s tumor-associated herpes-type virus to infectious mononucleosis. Proe. Natl. Aead.Sei. USA 59:94-101 19. Epstein, M. A., Achong,B. G., eds. 1979. The Epstein-BarrVirus. Berlin/ Heidelberg:Springer-Verlag.459 pp. 20. Henle, W., Diehl, V., Kohn,G., ZurHausen, H., Henle, G. 1967. Herpestype virus and chromosome marker in normal leukocytes after growth with irradiated Burkitt cells. Science 157: 1064-65 21. DeTbe’,G., Zeng, Y. 1982. Advances in ComparativeLeukemiaResearch,ed. D. S. Yohn,J. R. Blakeslee, pp. 00000. NewYork: Elsevier Biomedical 22. Miller, G., Shope,T., Lisco, H., Stitt, D., Lipman, M. 1972. Epstein-Barr virus: Transformation, cytopathic changesand viral antigens in squirrel monkey and marmoset leukocytes. Proe. Natl. Aead. Sei. USA69:383-87 23. Purtillo, D. T., Cassel, C., Yang,J. 1975. X-linked recessive progressive combinedvariable immunodeficiency (Duncan’sDisease). Lanceti: 935-41 24. Yarchoan,R., Redfield, R. R., Broder, S. 1986. Mechanismsof B cell activation in patients with acquired immunodeficiencysyndromeandrelated dis-

orders. Contributionof antibody-producingBcells, of Epstein-Barrvirus infected B cells, and of immunoglobulin production induced by human T cell lymphotropicvirus, type III/ lymphoadenopathy-associatedvirus. J. Clin. Invest. 78:439-47 25. Tosato, G., Blaese, R. M. 1985. Epstein-Barr infection and immunoregulation in man.Adv. Immunol.37: 99-149 26. Alspaugh,M. A., Henle, G., Lennette, E. T., Henle,W.1981.Elevatedlevels of antibodies to Epstein-Barr virus antigensin sera andsynovial tluids of patients with rheumatoidarthritis. J. Clin. lnvest. 67:1134-40 27. Ferrell, P. B., Aitcheson, C. T., Pearson,G. R., Tan, E. M.1981. Seroepidemiologicalstudy of relationships betweenEpstein-Barr virus and rheumatoidarthritis, d. Clin. lnvest. 67: 681-87 28. Martinez-Maza,O., Britton, S. 1983. Frequencies of the separate humanB cell subsets activatible to Ig secretion by Epstein-Barr virus and pokeweed mitogen.,/. Exp. Med.157:1808-14 29. Bird, G., Britton, S. 1979. A new approachto the study of humanB lymphocyte function using an indirect plaque assay, lmmunol.Reo. 45: 4152 30. Rosen, A., Gergely, P., Jondal, M., Klein, G., Britton, S. 1977.Polyclonal Ig productionafter Epstein-Barrvirus infection of humanlymphocytes in vitro. Nature267:52-54 31. Kirchner, H., Tosato, G., Blaese, R. M., Broder, S., Magrath,I. T. 1979. Polyclonal immunoglobulinsecretion by humanB lymphocytes exposed to Epstein-Barrvirus in vitro. J. lmmunol. 122:1310-13 32. Tosato, G., Blaese, R. M., Yarchoan, R. 1985. Relationship betweenimmunoglobulin production and immortalization by Epstein-Barr virus. J. Immunol. 135:959-64 33. Tosato, G., Magrath,I. T., Blaese, R. M. 1982. T Cell-mediated immunoregulation of Epstein-Barr virus (EBV) induced B lymphocyte activation in EBV-seropositive and EBV-seronegative individuals. J. Immunol.128: 575-79 34. Nemerow,G. R., McNaughton,M. E., Cooper, N. R. 1985. Binding of monoclonal antibody to the Epstein-Barr virus (EBV)CR2receptor induces activation and differentiation of humanB lymphocytes.J. Immunol.135:3068-73 35. Jondal, M., Klein, G. 1973. Surface

Annual Reviews

Annu. Rev. Immunol. 1988.6:85-113. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

IMMUNOBIOLOGY OF CR2 markers on humanB and T lymphocytes. J. Exp. Med.138:1365-78 36. Nemerow, G. R., Wolfert, R., McNaughton,M. E., Cooper, N. R. 1985. Identification and characterization of the Epstein-Barrvirus receptor on humanB lymphocytes and its relationship to the C3dcomplement receptor (CR2).J. Virol. 55:347-51 37. Fingeroth, J. D., Weis,J. J., Tedder, T. F., Strominger,J. L., Biro, P. A., Fearon, D. T. 1984. Epstein-Barrvirus receptor of humanB lymphocytes is the C3dreceptor CR2.Proc. Natl. Acad. Sci. USA81:4510-14 38. Simmons, J. G., Hutt-Fletcher, L. M., Fowler,E., Feighny,R. J. 1983.Studies of the Epstein-Barr virus receptor foundon Raji ceils. J. Immunol.130: 1303-8 39. Ross,G. D., Polley, M.J., Rabellino, E. M., Grey,H. M.1973. Twodifferent complementreceptors on humanlymphocytes.Onespecific for C3bandone specific for C3binactivator-cleaved C3b. J. Exp. Med.138:798-811 40. Eden,A., Miller, G. W., Nussenzweig, V. 1973. Humanlymphocytes bear membrane receptors for C3band C3d. J. Clin. lnvest. 52:3239-42 41. Okada, H., Nishioka, K. 1973. Complement receptors on cell membranes. J. Immunol. 111:1444-49 42. Nadler, L. M., Stashenko, P., Hardy, R., van Agthoven,A., Terhorst, C., Schlossman, S. F. 1981. Characterization of a humanB cell specific antigen (B2) distinct fromB1. J. Immunol. 126:1941-47 43. Slaw, M. F. E., Nemerow,G. R., Cooper, N. R. 1986. Biochemicaland antigenic analysis of the Epstein-Barr virus/C3dreceptor (CR2).J. Immunol. 136:4146-51 44. Wilson,B. S., Platt, J. L., Kay,N. E. 1985. Monoclonalantibodies to the 140,000molwt glycoprotein of B lymphocyte membrane(CR2 receptor) initiates proliferationof Bceils in vitro. Blood 66:824-29 45. Cohen,J. H. M., Fischer, E., Kazatchkine, M.D., Brochier, J., Revillard, J. P. 1986. Characterization of monoclonal antihuman-B cell antibody BLI3 as an anti-C3d receptor (CR2) antibody. Scand. J. Immunol.23:279-85 46. Campana,D., Janossy, G., Bofill, M., Trejdosiewicz, L. K., Ma, D., Hoffbrand, A. V., Mason,D. Y., Lebaco, A. M., Forster, H. K. 1985. Human B cell development.J. lmmunol.134: 1524-30 47. Rao, P. E., Wright, S. D., Westberg,

109

E. F., Goldstein, G. 1985. OKB7,a monoclonalantibody that reacts at or near the C3d binding site of human CR2. Cell. Immunol.93:549-55 48. Frade, R. 1986. Structure and functions of gpl40, the C3d/EBV receptor (CR2) of human B lymphocytes. Molec. Immunol.23:1249-53 49. Micklem,K. J., Sim, R. B., Sim, E. 1984. Analysisof C3receptor activity on humanB lymphocytesand isolation of the complementreceptor type 2 (CR2). Biochem.J. 224:75-86 50. Nemerow,G. R., Slaw, M. F. E., Cooper,N. R. 1986.Purification of the Epstein-Barr virus/C3d complement receptor of human B lymphocytes: Antigenicand functional properties of the purified protein. J. Virol. 58: 70912 51. Weis,J. J., Richards,S. A., Smith,J. A., Fearon,D. T. 1986. Purification of the B lymphocytereceptor for the C3d fragment of complementand the Epstein-Barr virus by monoclonalantibodyaffinity chromatography and assessment of its functional capacities. J. Imrnunol. Methods 92:79-87 52. Lambris, J. D., Dobson,N. J., Ross, G. D. 1981. Isolation of lymphocyte membranecomplementreceptor type two (the C3dreceptor) and preparation of receptor-specific antibody. Proc. Natl. Acad. Sci. USA78:1828-32 53. Frade, R., Bard, M., Ehlin-Henriksson, B., Klein, G. 1985.gpl40, the C3d receptor of humanB lymphocytes, is also the Epstein-Barrvirus receptor. Proc. Natl. Acad. Sci. USA82: 149093 54. Magrath,I., Freeman,C., Santaella, M., Gadek,J., Frank, M., Spiegel, R., Novikovs,L. 1981. Induction of complementreceptorexpressionin cell lines derived from humanundifferentiated lymphomas.J. Immunol. 127: 103943 55. Yefenof,E., Klein, G. 1977. Membrane receptor stripping confirmsthe association between EBVreceptors and complementreceptors on the surface of humanB lymphoma lines, lnt. J. Cancer 20:347-52 56. Rosenthal,K. S., Yanovich,S., Inbar, M., Strominger, J. L. 1978. Translocation of a hydrocarbonfluorescent probe betweenEpstein-Barr virus and lymphoidcells: An assay for early events in viral infection. Proc.Natl. Acad. Sci. USA75:5075-80 57. Hutt-Fletcher, L. M., Fowler,E., Lambris, J. D., Feighny,R. J., Simmons, J. G., Ross, G. D. 1983. Studies of the

Annual Reviews

Annu. Rev. Immunol. 1988.6:85-113. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

1 l0

COOPER~ MOORE& NEMEROW

Epstein-Barr virus receptor found on Raji cells. J. Immunol.130:1309-12 58. Tedder,T. F., Fearon,D. T., Gartland, G. L., Cooper,M.D. 1983. Expression of C3breceptors on humanB cells and myelomonocytic cells but not natural killer cells. J. Immunol.130:1668-73 59. Tedder,T. F., Weis,J. J., Clement,L. T., Fearon, D. T., Cooper,M. D. 1986. The role of receptors for complement in the induction of polyclonal B cell proliferation and differentiation. J. Clin. Immunol.6:65-73 G. 60. Mold,C., Cooper,N. R., Nemerow, R. 1986.Incorporationof the purified Epstein-Barrvirus/C3d receptor (CR2) into liposomes and demonstration of its dual ligand binding functions. J. Immunol. 136:4140-45 61. Wells,A., Koide,N., Stein, H., Gerdes, J., Klein, G. 1983. The Epstein-Barr virus receptor is distinct fromthe C3 receptor. J. Gen.Virol. 64:449-53 62. Inada, S., Brown,E. J., Gaither, T. A., Hammer, C. H., Takahashi, T., Frank, M. M. 1983. C3d receptors are expressed on humanmonocytesafter in vitro cultivation. Proc.Natl. Acad.Sci. USA80:2351-55 63. Vik, D. P., Fearon, D. T. 1985. Neutrophils express a receptor for iC3b, C3dgand C3dthat is distinct from CR1,CR2and CR3.J. Immunol. 134: 2571-79 64. Frade, R., Myones,B. L., Barel, M., Krikorian, L., Charriaut, C., Ross,G. D. 1985. gpl40, a C3bbinding membrane componentof lymphocytes, is the B cell C3dg/C3dreceptor (CR2) and is distinct from the neutrophil C3dgreceptor (CR4). J. Immunol.15: 1192-97 65. Shore, A., Dosch,H. M., Gelfand, E. W. 1979. Expression and modulation of C3 receptors during early T cell ontogeny. Cell. lmmunol.45:157~56 66. Patel, P., Menezes,J. 1981. EpsteinBarr virus (EBV)-lymphoid cell interactions. J. Gen.Virol. 53:1-11 67. Klein, G., Giovanella, B., Westman, A., Stehlin, J., Mumford, D. 1976. An EBVgenomenegative cell line established from an AmericanBurkitt lymphoma,lntervirology 5:319-34 68. Nilsson, K., Klein, G. 1982. Phenotypic and cytogeneticcharacteristics of humanB lymphoidcell lines and their relevancefor the etiology of Burkitt’s lymphoma.Adv. CancerRes. 37: 31980 69. Klein, G., Ehlin-Henriksson,B., $chlossman,S. F. 1983. Induction of an activated B lymphocyteassociated sur-

face moiety defined by the B2 monoclonal antibody by EBVconversion of an EBV-negative lymphomaline (Ramos):Differential effect of transforming (B95-8) and nontransforming (P3HR-1)EBVsubstrains. J. Immunol. 130:2985-89 70. Bofill, M., Janossy, G., Janossa, M., Burford, G. D., Seymour, G. J., Wernet, P., Kelemen,E. 1985. Human B cell development.J. Immunol.134: 1531-38 71. Hansson, M., Falk, K., Ernberg, I. 1983. Epstein-Barr virus transformation of humanpre-Bcells. J. Exp. Med. 158:616-22 72. Katamine, S., Otsu, M., Tada, K., Tsuchiya, S., Sato, T., Ishida, N., Honjo,T., Ono,Y. 1984. Epstein-Barr virus transformsprecursorB cells even before immunoglobulin gene rearrangements. Nature 309:369-72 73. Hibi, T., Chan, M. A., Petsche, D., Dosch, H. M. 1986. Phenotype, frequency, and EBVresponsiveness of humanmarrowB and pre-B cells. J. Immunol. 136:3211-18 74. Fu, G. M., Hurley, J. N., McCune,J. M., Kunkel,H. G., Good,R. A. 1980. Pre-Bcells and other possible precursor lymphoidcell lines derived from patients with X-linked agammaglobulinemia. J. Exp. Med.152:1519-26 75. Yarchoan,R., Tosato, G., Blaese, R. M., Simon,R. M., Nelson,D. L. 1983. Limitingdilution analysis of EpsteinBarr virus induced immunoglobulin production by humanB cells. J. Exp. Med. 157:1-14 76. Bird, A. G., Britton, S., Ernberg,I., Nilsson, K. 1981. Characteristics of Epstein-Barrvirus activation of human B lymphocytes.J. Exp. Med.154: 83239 J. F., Klein, G., Ernberg,I., 77. Schwaber, Rosen, A., Lazarus, H., Rosen,F. S. 1980.Deficiencyof Epstein-Barrvirus (EBV) receptors on B lymphocytes from certain patients, with common varied agammaglobulinemia.J. Immunol. 124:2191-96 78. Wang,F., Hutt-Fletcher,L., Blaese, R. M., Tosato, G. 1985. Epstein-Barr virus (EBV)binding is independent C3dreceptor expression.Fed. Proc.44: 1298(Abstr.) 79. Dosch,H. M., Chan,M., Stein, L. D., Hibi, I. 1985.Expressionof EBV receptors on EBVtransformable humanB cells. Fed. Proc.44:1287(Abstr.) 80. Aman,P., Ehlin-Henriksson,B., Klein, G. 1984. Epstein-Barr virus susceptibility of normal humanB lympho-

Annual Reviews

Annu. Rev. Immunol. 1988.6:85-113. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

IMMUNOBIOLOGY OF CR2 cyte populations. J. Exp. Med. 159: 208-20 81. Robinson, J., Frank, A., Henderson, E., Schweitzer,J., Miller, G.1979.Surface markersandsize of lymphocytes in humanumbilical cord blood stimulated into deoxyribonucleic acid synthesis by Epstein-Barrvirus. Infect. Immun.26: 225-31 82. Roome,A. J., Reading, C. L. 1987. Frequencyof B lymphocytetransformation by Epstein-Barr virus decreases withentry into the cell cycle, lmrnunol. 60:195-201 83. Jarvis, J. E., Ball, G., Rickison,A. B., Epstein, M.A. 1974.Cytogeneticstudies on humanlymphoblastoid cell lines from Burkitt’s lymphomasand other sources. Int. J. Cancer14:716-21 84. Nilsson, K., Andersson, L. C., Gahmberg,C. G., Wigzell, H. 1977. Surface glycoproteinpatterns of normal and malignant humanlymphoid cells. Int. J. Cancer20:708-16 85. Yarchoan,R., Engler, R. J., Barrow, L. A., Kurman,C. C., Nelson, D. L. 1984. Antigen-stimulatedhumanB cell clones are committedto the production of a single class of antibody in vitro. J. Immunol. 132:2696-99 86. Nunez,D., Charriaut-Mariangue, C., Barel, M., Benveniste, J., Frade, R. 1987. Activation of humanplatelets through gpl40, the C3d/EBV receptor (CR2).Eur. J.Immunol.17:515-20 87. Yu, G, H., Holers, V. M., Seya, T., Ballard, L., Atkinson, J. P. 1986. Identifications of a third component of complementbinding glycoprotein of humanplatelets. J. Clin. Invest. 78: 494-501 88. Bhan,A. K., Nadler, L. M., Stashenko, P., McCluskey,R. T., Schlossman,S. F. 1981.Stagesof B cell differentiation in humanlymphoidtissue. J. Exp. Med. 154:737-49 89. Stashenko, P., Nadler, L. M., Hardy, R., Schlossman,S. F. 1981.Expression of cell surface markersafter humanB lymphocyte activation. Proc.Natl. Acad. Sei. USA78:3848-52 90. Wolf, H., ZurHausen,H., Becker, V. 1973. EBviral genomesin epithelial nasopharyngealcarcinomacells. Nature 255:245-49 91. Huang,D. P., Ho, J. H. C., Henle,W., Henle, G. 1974. Demonstration of Epstein-Barrvirus associated nuclear antigen in nasopharyngealcarcinoma cells fromfresh biopsies.Int. J. Cancer 14:580-88 92. Glaser, R., Lang, C. M., Lee, K. J., Schuller, D. E., Jacobs, D., McQuattie,

1 1 1

C. 1980. Attempt to infect nonmalignant nasopharyngeal epithelial cells from humans and squirrel monkeys with Epstein-Barrvirus. J. Natl. Cancer Inst. 64:1085-90 93. Shapiro, I. M., Volsky, D. J. 1983. Infection of normalhumanepithelial cells byEpstein-Barrvirus. Science219: 1225-28 94. Sixbey,J. W.,Vesterinen, E. H., Nedrud, J. G., Rabb-Traub, N., Walton, L. A., Pagano,J. S. 1983.Replication of Epstein-Ban" virus in htmaanepithelial cells infected in vitro. Nature 306:480-83 95. Young,L. S., Sizbey,J. W.,Clark, D., Rickinson, A. B. 1986. Epstein-Barr virus receptors on humanpharyngeal epithelia. Lanceti: 240-42 96. Kehrl, J. H., Muraguchi, A., Butler, J. L., Falkoff,R. J. M.,Fauci, A.S. 1984. Human B cell activation, proliferation and differentiation. Immunol.Rev. 78: 75-96 97. Howard,M., Nakanishi, K., Paul, W. E. 1984. B cell growth and differentiation factors. Immunol.Rev. 78: 185-210 98. Kishimoto,T., Yoshizaki,K., Kimoto, M., Okada,M., Kuritani, T., Kikutani, H., Shimizu, K., Nakagawa,T., Nakagawa,N., Miki, Y., Kishi, H., Fukunaga, K., Yoshikubo, T., Taga, T. 1984.B cell growthanddifferentiation factors and mechanism of B cell activation. Immunol.Rev. 78:97-118 99. Jelinek, D. F., Lipsky, P. E. 1987. Regulation of humanB lymphocyte activation, proliferation and differentiation. Adv. Immunol.40:1-59 100. Tosato, G., Marti, G. E., Yarchoan, R., Heilman,C. A., Wang,F., Pike, S. E., Korsmeyer,S. J., Siminovitch,K. 1986. Epstein-Barrvirus immortalization of normalcells of Bcell lineage with nonproductive,rearranged immunoglobulin genes. J. Immunol. 137: 2037-42 101. Boyd,A. W., Anderson,K. C., Freedman,A. S., Fischer, D. C., Slaughenhoupt,B., Schlossman,S, F., Nadler, L. M.1985. Studies of in vitro activation and differentiation of humanB lymphocytes.J. Immunol.134:1516-23 102. Gordon, J., Walter, L., Guy, G., Brown,G., Rowe,M., Rickinson, A. 1986. Control of humanB lymphocyte replication. Immunol.58:591-95 103. Weis, J. J., Fearon, D. T, 1985. The identification of N-linkedoligosaccharides on the humanCR2/EpsteinBarr virus receptor and their function in receptor metabolism, plasma mem-

Annual Reviews

Annu. Rev. Immunol. 1988.6:85-113. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

112

COOPER, MOORE& NEMEROW

brane expression and ligand binding. d. Biol. Chem.260:13824-30 104. Reinherz,E. L., Haynes,B. F., Nadler, L. M., Bernstein,J. D., eds. 1986.LeukocytetypingII. In Proeeedin~Ts of the Second International Workshop on Human LeukocyteDifferentiation Antigen. NewYork: Springer-Verlag. 549 PP. 105. Weis,J. J., Fearon,D. T., Klickstein, L. B., Wong,W.W., Richards, S. A., deBruynKops, A., Smith, J. A., Weis, J. H. 1986.Identification of a partial cDNAclone for the C3d/Epstein-Barr virus receptor of humanB lymphocytes: Homologywith the receptor for fragmentsC3band C4bof the third and fourth components of complement. Proc. Natl. Acad. Sci. USA 83:5639-43 106. Reid, K. B. M., Bentley, D. R., Campbell,R. D., Chung,L. P., Sim,R. B., Kristensen, T., Tack, B. F. 1986. Complementsystem proteins which interact with C3bor C4b. Immunol. Today 7:230-34 107. Leonard, W. J., Depper, J. M., Kanehisa,M., Kronke,M., Peffer, N. J., Svetlik, P. B.~Sullivan,M., Greene, W.C. 1985. Structure of the human interleukin-2 receptor gene. Science 230:633-39 108. Klickstein, L. B., Wong,W.W.,Smith, J. A., Weis, J. H., Wilson, J. G., Fearon, D. T. 1987. HumanC3b/C4b receptor (CR1).Demonstrationof long homologousrepeating domains that are composedof the short consensus repeats characteristic of C3/C4binding proteins. J. Exp. Med.165:1095-1112 109. Weis,J. H., Morton,C. C., Bruns, G. A. P., Weis,J. J., Klickstein, L. B., Wong,W.W., Fearon, D. T. 1987. A complement receptor locus: Genes encoding C3b/C4b receptor and C3d/Epstein-Barr virus receptor map to Iq32. J. lmmunol.138:312-15 110. Chan, M. A., Stein, L. D., Dosch, H. M., Sigal, N. H. 1986. Heterogeneityof EBVtransformable human B lymphocytepopulations. J. Immunol.136: 106-12 111. Ho, C., Aman,P., Masucci,M., Klein, E., Klein, G. 1986.Bcell activation by the nontransforming P3HR-1substrain of the Epstein-Barrvirus (EBV). Eur. J. Immunol.16:841-45 112. Hutt-Fletcher, L. M. 1987.Synergistic activationof cells by Epstein-Barr virus andB cell growthfactor. J. Virol. 61: 774-81 113. Melchers, F., Erdel, A., Corbel, C., Leptin, M., Schulz, T., Dierich, M.P.

1986. Cell cycle control of activated, synchronizedmurine B lymphocytes-roles of macrophagesand complement C3. Molec. Immunol.23:1173-76 114. Frade, R., Crevon,M. C., Barel, M., Vazques,A., Krikorian, L., Charriaut, C., Galanaud, P. 1985. Enhancement of humanB cell proliferation by an antibodyto the C3dreceptor, the gp 140 molecule.Eur. J. Immunol.15:73-76 115. Mitler, R. S., Talle, M.A., Carpenter, K., Rao, P. E., Goldstein, G. 1983. Generation and characterization of monoclonalantibodies reactive with humanB lymphocytes. J. Immunol. 131:1754-61 116. Changelian,P. S., Fearon, D. T. 1986. Tissuespecific phosphorylationof complement receptors CR1and CR2. J. Exp. Med. 163:101-15 117. Barel, M., Vazquez,A., Charriaut, C., Aufredou,M. T., Galanaud,P., Frade, R. 1986. gpl40, the C3d/EBV receptor (CR2),is phosphorylateduponin vitro activation of humanperipheral B lymphocytes. FEBSLett. 197:353-56 118. Nemerow, G. R., Cooper, N. R. 1984. Early events in the infection of human B lymphocytesby Epstein-Barr virus: Theinternalization process. Virology 132:186-98 G. R., Cooper, N. R. 1984. 119. Nemerow, Infection of B lymphocytesby a humanherpesvirus,Epstein-Barrvirus, is blocked by calmodulin antagonists. Proc. Natl. Acad. Sci. USA81: 495559 120. Cambier,J. C., Ransom,J. T. 1987. Molecular mechanismsof transmembranesignaling in B lymphocytes.Ann. Rev. Immunol.5:175-99 121. Meuth,J. L., Morgan,E. L., DiScipio, R. G., Hugli, T. E. 1983. Suppression of T lymphocytefunctions by human C3fragments. J. Imrnunol.130: 260511 122. Thoman,M. L., Meuth,J. L., Morgan, E. L., Weigle,W.O., Hugli,T. E. 1984. C3d-K,a kallikrein cleavage fragment of iC3bis a potent inhibitor of cellular proliferation. J. Immunol.133: 262933 123. Hobbs,M.V., Feldbush,T. L., Needleman,B. W., Weiler, J. M. 1982. Inhibition of secondaryin vitro antibody responses by the third componentof complement.J. Immunol.128:1470-75 124. Pepys, M.B. 1972. Role of complement in inductionof the allergic response. Nature 237:157-59 125. Pepys, M.B. 1974. Role of complement in induction of antibodyproductionin vivo. J. Exp. Med.140:126-45

Annual Reviews

Annu. Rev. Immunol. 1988.6:85-113. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

IMMUNOBIOLOGY OF CR2 126. Dukor, P., Schumann,G., Gisler, R. H., Dierich, M., Konig,W., Hadding, U., Bitter-Saermann, D. 1974. Complement dependentB cell activation by cobra venomfactor and other mitogens. J. Exp. Med.139:337-54 127. Romball, C. G., Ulevitch, R. J., Weigle, W.O. 1980. Role of C3 in the regulationof a splenic PFCresponsein rabbits. J. Immunol.124:151-55 128. Pryjma,J., Humphrey, J. H., Klaus, G. G. 1974. C3 activation and T independentBcell stimulation. Nature252: 505-56 129. Pryjma,J., Humphrey, J. H. 1975. Prolonged C3 depletion by cobra venom factor in thymus-deprived miceandits implication for the role of C3as an essential secondsignal for Bcell triggering. Immunology28:569-76 130. Klaus, G. G., Humphrey,J. H. 1977. Thegeneration of memory cells. Immu-

113

nology 33:31-40 131. Thorbecke,G. J., Romano,T. J., Lerman, S. P. 1974. Regulatory mechanismsin proliferation anddifferentiation of lymphoidtissue, with particular referenceto germinalcentredevelopment. Prog. Immunol.3:25-34 132. Klaus, G. G., Humphrey, J. H., Kunkl, A., Dongworth,D. W.1980. The follicular dendriticcell: Its role in antigen presentation in the generation of immunological memory.Immunol. Rev. 53:3-28 133. Mandel,I. E., Phipps,R. P., Abbot,A., Tew,J. G. 1980.Thefollicular dendritic cell: Longterm antigen retention during immunity, lmmunol.Rev. 53: 2959 134. Klaus, G. G. B., Humphrey, J. H. 1986. A re-evaluation of the role of C3in B cell activation. Immunol.Today7: 16365

Annu. Rev. Immunol. 1988.6:85-113. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Annu. Rev. Immunol. 1988.6:85-113. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Annual Reviews www.annualreviews.org/aronline Ann. Rev. lmmunol. 1988. 6:115-37 Copyright © 1988 by Annual Reviews lnc. All rights reserved

VETO CELLS Annu. Rev. Immunol. 1988.6:115-137. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Pamela J. Fink Departmentof Biology, University of California at San Diego, La Jolla, California 92093 Richard P. Shimonkevitz

and Michael J. Bevan

Departmentof Immunology, ResearchInstitute of Scripps Clinic, 10666North Torrey Pines Road, La Jolla, California 92037 INTRODUCTION One of the main problems confronting an immunesystem designed to eliminate foreign substancesis the establishmentand maintenance of selftolerance. Twoschoolsof thoughtexist on howself-reactivity in the T cell compartmentis avoided. Onemechanism,clonal deletion, proposes that self-tolerance is imposedon immatureT cells in the thymus--that at a certain stage of differentiation, contact with antigen permanentlyinactivates the cell (1, 2). Analternative, nonmutually exclusivesuggestion that anti-idiotypic suppressormechanisms exist to inhibit continually any potential self reactivity. Accordingto this hypothesis, anti-A reactive T cells are held in checkby anti-(anti-A) suppressorT cells (3, 4, 5). self-tolerance in the populationof T cells is apparentlyimposedby clonal deletion of autoreactive lymphocyteswithin the thymus. However,this mechanism of avoiding autoimmune recognition mayalone be insufficient, as autoreactive T cells can be isolated from the population of mature peripheral lymphocytes,and in fact, syngeneiccytolysis can be demonstrated in manyallospecific cytotoxic T lymphocyte(CTL)clones (6). In the following pages weargue for the presence of a mechanism,other than one involvinganti-idiotypic networks,for the continuedmaintenance of self-tolerance in the pool of peripheral lymphocytes.This form of antigen-specific suppressionresults in the functional eliminationof autoreactive peripheral T cells by other lymphoid cells, the mosteffective being 115 07324582/88/0410-0115502.00

Annual Reviews

Annu. Rev. Immunol. 1988.6:115-137. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

116

FINK,

SHIMONKEVITZ & BEVAN

T cells. However,in this interaction, the antigen-specific receptor of the "suppressor cell" is not involved. The cells responsible for maintainingself-tolerance in this waywere first named "veto cells" by Miller and coworkers because they apparently functioned by exercising the power of veto over T cell immuneresponses; a T cell deemed autoreactive by a veto cell was quickly rendered nonfunctional (7). Because vetoing seems to result from a nonimmunogenic, suppressive interaction, it is possible that any cell type expressing antigen (a fibroblast, for example) could serve as a veto cell. However,in vivo, lymphoid cells are muchmore likely to comeinto direct contact with and to present antigen to other T lymphocytesthan are fibroblasts. The in vivo definition of an effective veto cell maytherefore be very different from an in vitro definition. Althoughthe apparent lineage of the veto cell and the duration and severity of the ensuring hyporeactivity differ somewhatwith differing experimental protocols, perhaps a single property defines veto cell-induced tolerance. That property is the unidirectional recognition of the veto cell by the future ~’victim" of that veto cell (8). By recognizing antigen on the surface of a veto cell, a T cell defines itself as autoreactive in the eyes of the veto cell. The veto cell, in the absence of any receptors specific for the autoreactive cell, renders that boundT cell nonfunctional. The specificity of this type of tolerance induction resides only in the cell to be tolerized, and "self" is defined by the antigens expressed on the veto cell surface. This unidirectional recognition event is schematizedin Figure 1. The simplicity and economyof this type of tolerance mechanismshould be readily apparent. There is no requirement for a specialized lineage of cells bearing diverse antigen specific receptors. Tolerance is instead maintained by cells with other immuneroles, including that of antigen specific, majdr histocompatibility complex(MHC)-restricted cytotoxicity. In other words, CTLthat can rid the body of virus infected cells can also function to maintain self-tolerance (9). This system is not only simple, is efficient, ensuring that the autoreactivity frequently generated during the course of a specific immuneresponse will be immediately squelched by the very cells mountingthat specific response (10). Wenow examinethe data pointing to the existence of veto cell-induced tolerance, beginning with the experiments of the main proponents of this mechanism,and following with the reinterpretation of other experiments in light of this modelfor the maintenanceof self-tolerance.

VETO CELLS CAN INACTIVATE CTL PRECURSORS Experiments directed primarily at studying veto cells have focussed on their ability to suppress specifically any CTLprecursors directed against

Annual Reviews VETO CELLS

117

Annu. Rev. Immunol. 1988.6:115-137. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

iEl’OCELL

Figure 1 Unidirectional recognition is the basis for veto cell function. Anautoreactive T cell recognizes the self antigens of the veto cell and is in turn suppressed. The antigen specific receptors of the veto cell are not engaged.

them, whetherthose CTLbe alloreactive, recognizingthe veto cell MHC, or MHC restricted, recognizingveto cell-expressed haptenor minorhistocompatibility(H) antigens. Initial experiments all utilized an in vitro system for the induction and suppressionof CTL,while the possible physiological role of veto cell mediatedsuppressionis basedon the morerecent in vivo experiments. In Vitro Experiments BONE MARROW--DERIVED T~CELL COLONIES Miller and his coworkersbegan their studies of veto cell-mediated suppressionwith the discovery that spleen cells from athymic (nude) mice can partially suppress a primary CTLresponse against allogeneic MHC antigens (11). The experimental protocol called for the coculture of lymphnode responder cells with irradiated allogeneic stimulators and graded doses of irradiated nude spleencells syngeneicwith the stimulator cells in a 5-daymixedlymphocyte culture (MLC).In contrast to the situation with nudemice, spleen cells from normal, euthymicmice did not suppress. However,thymocytesand bone marrowcells from euthymic mice were found to suppress a CTL responsedirected against their MHC antigens, and this suppression(unlike that mediatedby nudespleencells) wassensitive to 7 irradiation (11, 12). Non-MHC genes appear to influence the strength of veto cell-mediated suppressionby normalbone marrowcells (13).

Annual Reviews 118

FINK, SI-tIMONKEVITZ & BEVAN

strain D lymph node ceils ÷ ~stroin A spleen cells Assayfor lysis of strain A targets

~ OOo "~ 5d

Annu. Rev. Immunol. 1988.6:115-137. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

strain A bone methylceilulose, marrow cells conditioned medium

strainAmspleencells

strainA spleen cells primedto Am + ~ stra|nAmspleencells Assayfor

~

3d

(’~

Co

CTLClone

~

Lymph node responders

(+) k

5d ~ Assay for

lysls and strain C targets

strain A

(+) S~at~s

}

FixTure 2 In vitro systems for measuring the inactivation of CTLprecursors by veto cells. (a) T cell colonies derived from strain Abone marrowcells can suppress anti-A specific CTL. A spleen cells can suppress anti-A m CTL. (c) A CTL (b) Cultured, minor H bearing, MHC clone expressing irrelevant T cell receptors can suppress the CTLresponse directed against its MHCantigens.

The level of specific suppression in this in vitro system remained unimpressive, however,until it was discovered that this novel type of suppressor cell activity is enriched 30-100-fold on a per cell basis in T cell colonies grown from bone marrow cells from normal mice (see Figure 2a). These

Annual Reviews

Annu. Rev. Immunol. 1988.6:115-137. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

VETO CELLS

119

colony-derived suppressors were found to be resistant to ~ irradiation but sensitive to UVirradiation (14). Data indicated these T cells originate from Thy-1 negative, radiosensitive precursors found in the bone marrow but not in the spleens of normal mice (12). Lymphoidcolonies grown from fetal liver cells from normal mice also demonstrate some suppressive activity, although they cannot be classified as veto cells becauseof the lack of specificity for suppression of only those responses directed against antigens expressed by the fetal liver colonies (15). The results of these experiments can be summarizedas follows: Spleen and bone marrow cells from nude mice, and thymocytes, bone marrow cells, and T cell colonies grown from the bone marrowof normal mice can specifically suppress the primary CTLresponse directed against the MHC antigens expressed by these cells. Such veto cell activity is not exhibited by unmanipulated spleen or lymph node cells from normal mice. However, the experiments described below indicate that activated spleen cells from normal mice can serve as veto cells in vitro. PRECULTURED SPLENICT CELLSUnprimed spleen cells cultured in the absence of any knownexternal stimulation for 2-3 days can inhibit the generation of MHC-restricted CTLspecific for a minor H antigen expressed by those spleen cells (16). The experimental protocol used to assay this suppression is outlined in Figure 2b. Similarly, spleen cells cultured for 2 days in the presence of a T cell mitogen are able to suppress CTL responses directed against them, although with somewhatless specificity (17). Normal splenic responder cells harvested on days 3-5 of MLCcan also suppress the development of allogeneic CTLdirected against their MHCantigens (18). Within the cultured spleen cell population, CDSpositive T cells are largely responsible for this suppression (16). The degree of suppression in some experiments is impressive, being dose to 100%at a veto cell: responder cell ratio of 1 : 1 (16). Nosuppression is seen using either freshly explanted antigen-bearing spleen cells or cultured third party cells bearing minorH antigens other than those expressed by the stimulator cells (16). The suppression therefore showsthe antigen specificity of veto cell function. CLONED VETOCELLSThe in vitro study of veto cells, particularly the mechanism of their function, has profited from the use of a uniform population of potent veto cells. This was madepossible with the discovery that cloned T cells with cytolytic activity directed against irrelevant antigens can efficiently and specifically suppress the generation of CTLdirected against antigens they express (17, 19, 20). As shownin Figure 2c, to best assay this activity, a primary MLCis set up by coculturing strain B responders with equal numbers of irradiated A and C stimulator cells,

Annual Reviews

Annu. Rev. Immunol. 1988.6:115-137. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

120

FINK,

SHIMONKEVITZ & BEVAN

where A, B, and C are mousestrains differing at the MHC.At the end of 5 days, CTLactivity is measured, revealing approximately equal and efficient lysis by the strain B effector cells of target cells bearing A or C antigens. If small numbers of cloned anti-D.specific CTLbearing A antigens are added into this MLCon day 0, at the end of 5 days, the anti-C CTLactivity from the surviving responder cells is undiminished, while the anti-A activity is greatly reduced (17, 19). This suppression is mediated cloned CTLin the absence of any antigen they can recognize and is not only highly specific but extremely potent--a 50- to 100-fold reduction in specific CTLactivity is mediated by veto cells seeded at 1/100 the initial numberof responder cells. In all, 8/8 CTLclones (7 alloreactive and MHCrestricted) exhibited veto activity, indicating that suppression of anti-self responses is one of the normal functions of CTL(17, 20). In good agreement with other work (16), this effect is radiation sensitive and property only of CD8-bearingT cell clones (17). HOWDO VETO CELLS FUNCTIONIN VITRO? The use of the

cultures

described

above--containing two types of stimulator cells and one type of veto cell--eliminates competition for nutrients and overcrowding as possible explanations for howveto cells suppress (17, 19). Furthermore, addition of growth factor-containing supernatants does not override the veto, indicating that veto cells neither prevent production of helper factors nor function merely as highly localized lymphokinesinks (17). Veto cells not function simply as unlabeled competitors for responder cell attention during the CTLassay. This is dear because removingveto cells on the day of assay from a culture by antibody-plus-complement treatment does not abrogate the suppression (17, 20). Furthermore, mixing suppressed and nonsuppressedcultures just before assay does not result in suppression of the latter responders (20). Neither supernatants removedfrom cloned veto cells nor those removedfrom veto cell-suppressed cultures will diminish a CTLresponse (19). Thus, veto cell activity cannot be replaced by soluble factors, indicating that somekind of cell-cell contact is required. Onestudy using timed addition and removalof cloned veto cells from the cultures to be suppressed indicates that veto cells need to be present throughout the culture period, at both early and late timepoints (19). However, other experiments suggest that CTLprecursors are not susceptible to veto cell activity prior to 20 hr of stimulation (14, 20). A resolution to this disagreement is important for determining whether activation of CTLprecursors is a prerequisite for their suppression, a key point whenconsidering intrathymic tolerance induction (see below). Limiting dilution analyses indicate that veto cell suppression appears to operate through a decrease in the number of CTLprecursors and not a

Annual Reviews

Annu. Rev. Immunol. 1988.6:115-137. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

VETOCELLS

121

reduction in the cytotoxicity generated per precursor (12, 14). Reculturing responder cells separated from veto cells after 43 hr with fresh stimulator cells does not reverse their suppression, although only 4 days were allowed for this reversal (14). More long-term culturing of inactivated CTLprecursors in limiting dilution would be required to answer adequately whether or not veto cell suppression of CTLprecursors is reversible, and these experiments have yet to be reported. Separation of suppressed cultures on the basis of CD8expression and addition of these sorted cells to a fresh MLCindicates that veto cells do not induce CD8-positive suppressor cells within the responder population, which then mediate the veto cell function (14). Veto cells function in vitro through the type of unidirectional recognition diagrammedin Figure 1. Thus, cloned CTLexpressing irrelevant receptors are capable of vetoing CTLprecursors that recognize them. Furthermore, only haptenated nude spleen cells can veto hapten-specific CTLin a syngeneic system (21). These in vitro studies on the suppression of CTLresponses by veto cells have given us quite a bit of information on veto cell function. Weknow that veto cell-induced suppression is not due to overcrowding, growth factor deprivation, or cold target competition. Suppression requires both prolonged coculture of veto cell and responder cell populations and cell contact, and it does not operate through a CDS-posifive suppressor induced in the responder population. Suppression is highly specific; the specificity is defined by the antigens expressed on the veto cell surface and the CTLthat recognizes those antigens. Veto cells function directly by decreasing the number of CTLprecursors, and this inactivation is not readily reversed. The question of whether veto cells function by lysing CTLprecursors that recognize them is a more difficult one to answer and is discussed at length below. In Vivo Experiments Before assigning to veto cells any potential role in the maintenance of self-tolerance, it is essential to showconvincingly that veto cells function in vivo. There are two main systems in which suppression of CTLresponses by the veto mechanismhas been demonstrated in vivo, one involving suppression of a class I response (9, 22, 23) and the other, the suppression of an MHCrestricted, minor H antigen-specific response (16, 24-28). Webegin with the class I-specific response because it is analogousto the in vitro systems described above. Suppression of class I-specific CTLis initiated by the injection of splenic T cells intravenously into host mice differing from the injected cells at one or several class I loci. Five or six days later, host PROTOCOLS FOR ASSAYING CTL SUPPRESSION IN VIVO

Annual Reviews

Annu. Rev. Immunol. 1988.6:115-137. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

122

FINK, SHIMONKEVITZ & BEVAN

spleen cells were cocultured with irradiated stimulator cells bearing class I antigens shared by the injected spleen cells as well as additional class I antigens not sharedby either donoror host cells. After 5 days of culture, CTLactivity was measuredagainst targets bearing these shared or third party antigens. This experimentalprotocol is outlined in Figure 3a. In each of the several antigen combinationstested, a goodCTLresponse was elicited against the third party antigens, while the CTLresponseagainst those antigens shared by the injected spleen cells wassuppressed.As few as 2.5 × 106 injected splenic T cells mediateda significant (20-fold)class antigen-specific suppression (23). Specific unresponsivenesslasted for weeks,and in somecases, recoverywasstill incompleteafter 5 months(9). Suppressionof minorHantigen-specific CTLis inducedby injection of A~’ (minor H antigen-bearing, H-2A) spleen cells into F~(A× B) mice. CTLspecific for the injected minorH antigen in the context of H-2B are efficiently and rapidly induced in such animals by the phenomenon of "cross-priming,"presumablythe result of the processingof the injected antigen and presentation on host F~(A× B) antigen-presenting cells. H-2A-restricted CTLcan be inducedby either cross primingor by direct primingby the injected spleen cells themselves(29). Spleen cells from 7 10

spleen cells

strain AA splenic T cells

~strain AC spleen cells

~ ..~ ~ straln DO mouse

b.

spleen cells

10 7

~ m strain A spleen cells

~ ~

AB and BC Assa~lof l~lsis targets

vslpleen cells /J:L~ ~

~

Assay lgsis of Amandi~mtargets

FI (AxB) mouse Figure3 In viva induction of veto cell mediated suppression of CTLactivity. (a) Suppression ^ antigens into a strain of an anti-MHCresponse is induced by injecting cells bearing KAD c antigens. B mouse.Host spleen cells are cultured 6 days later with stimulators bearing KAD B and compared Suppression of the K^-specific response is measured on targets bearing KAD c targets. (b) Suppression of an antito a control response against Dc measured on K"D ^minor H specific, MHC restricted response is induced by injecting minor H different MHC (A~) cells into F~(A x B) hosts. Host spleen cells are cultured with minor H-bearing stimulator cells. Suppression of anti-A m CTLactivity is measured relative to the nonsuppressed, cross primed activity measuredon Bm targets.

Annual Reviews

Annu. Rev. Immunol. 1988.6:115-137. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

VETO

CELLS

123

F1(A× B) animals injected with as few as 107 viable m spleen c ells w ere cocultured 5q5 days later with irradiated FI(Am x Bm) stimulators, and another 5 days later CTLactivity directed against Am and Bm targets was measured. This revealed a suppression of the anti-A m CTLresponse (Figure 3b). In all the strain combinations tested, a dramatic skewing the ratio of direct versus cross-primed CTLactivity was seen, the result of a haplotype- and antigen-specific suppression of direct primed CTL activity (24). Althoughperhaps more complicated than the class I-specific CTLsuppression described above, this minor H-specific system includes the ideal internal control--an MHCrestricted CTLresponse induced by the injection of the same spleen cells and boosted by the same stimulator cells in the samecultures as those used to induce and boost the suppressed response. One of the distinguishing characteristics of this type of CTLsuppression is that it is both haplotype specific (Am killing is depressed, while Bmkilling is not) and antigen specific (Amkilling is depressed, but A-restricted, haptenspecific cytolysis is unaffected) (24). The extreme specificity of this suppression is in contrast to the generalized immunosuppression that is characteristic of graft-versus-host disease (30) or that is predicted by some anti-idiotypic suppressor hypotheses. Furthermore, the severity and the duration of the CTLhyperactivity induced by the injection of minor H different spleen cells is directly related to the numberof spleen cells injected. Injection of 107 Amspleen cells into F~(A× B) hosts results in a ratio Amto Bmkilling of 1 : 25 on day 4 after injection, tapering off to 1 : 1 at day 10, while injection of 6 x 107 Amcells results in a ratio of < 1:60 even at day 10. This temporarily skewed ratio of direct to cross-primed CTL activity is due to a depressed direct primed response rather than to a heightened cross-primed response for the following reasons. The crossprimed response is induced by F~ antigen presenting cells which should m equally well induce the direct primed response. In addition, irradiated A spleen cells prime both Am and Bm responses equally well, and with the same kinetics as the anti-Bmresponse induced by Am spleen cells. As with the in vitro induced suppression, this type of suppression is not easily reversible; boosting cells in culture for two rounds with irradiated stimulators does not appreciably alter the ratio of Amto Bmkilling (24). And finally, it is more difficult to suppress the Am CTLresponse in FI(A x B) mice already primed to the minor H antigens in question than it is to suppress the response in naive mice (24, 26). CHARACTERISTICS OF THE SPLEEN CELL--INDUCED SUPPRESSION

PROPERTIES OF THE CELLS RESPONSIBLE FOR CTL SUPPRESSION IN VIVO The spleen cells responsible for inducing CTLhyporeactivity in both the class I

Annual Reviews

Annu. Rev. Immunol. 1988.6:115-137. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

124

FINK,

SHIMONKEVITZ & BEVAN

and minor H-specific systems are Ia negative, radiosensitive, nylon wool nonadherent cells that are depleted (though not entirely) by treatment with anti-Thy-1 plus complement(16, 22-25). Within the population of splenic T cells, CD8positive cells are the most potent (by about 10-fold) inducing this suppression, with as few as 3 x 106 CD8positive, Thy-1 positive cells mediating a 20-fold suppression of specific CTLactivity (24). However, even Thy-1 negative spleen cells from nude mice can induce CTLsuppression, although inefficiently (23). The cells responsible for inducing CTLsuppression in vivo are present in low numbers in the thymus and bone marrowof normal mice (24) and are not present in skin grafts (28). Perhaps the most distinctive feature of the cells responsible for this type of CTLsuppression is that they are not required to recognize the cells they are suppressing. This conclusion derives from the following information. Spleen cells preprimed to antigens of the responder animal are not more efficient suppressors, on a per cell basis, than are naive spleen cells (24). Cells from [Am -~ Fl(Amx Bm)]radiation chimeras, that are tolerant of and B MHCantigens, are efficient at suppressing anti-A m CTLactivity from recipient F1(A x B) animals (24). In fact, in a system in which injected spleen cells can be separated at the time of culture from host cells on the basis of Thy-1 expression, it was apparent not only that the host cells are incapable of lysing donor cells, but that the donor veto cells are incapable of recognizing host cell antigens. In other words, there is mutual tolerization in animals injected with class I incompatible cells (31). Most conclusively, F~(Am x A) cells can suppress the anti-A TM CTLfrom A animals to which they are tolerant (24, 26). That foreign spleen cells can efficiently suppress a CTLresponse in animals to which they are tolerant is one of the primary reasons for categorizing this suppression as veto cellinduced. HOWDO VETO CELLS FUNCTION IN VIVO? The

suppression induced by

the

injection of antigen-bearingspleen cells is class I-restricted in the induction phase and cannot be overridden in vitro by growth factor-containing supernatants 06, 18, 23, 25). Both of these findings suggest that the suppression operates at the level of the CTLitself and not indirectly by inactivating helper cells required for the activation of CTL.In the case of the minor H system, such helper cells would need both to recognize minor H antigens in the context of MHC molecules and to be able to distinguish minor H-specific CTLrestricted to H-2A B. from those restricted to H-2 Only such a complicated recognition system could explain the specific inactivation by Am spleen cells of CTLspecific for minor H antigens plus H-2g. In fact, limiting dilution analyses of spleen cells from suppressed

Annual Reviews

Annu. Rev. Immunol. 1988.6:115-137. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

VETO CELLS

125

versus nonsuppressed animals indicate a greater than 200-fold reduction in the numberof CTLprecursors specific for the injected antigen, with no detectable change in the numberof CTLprecursors specific for third party antigens (23). Thus, spleen cell-induced suppression operates at the level of CTLprecursors in vivo just as it does in vitro, causing a functional clonal deletion of specific CTL. Is this reduction in CTLprecursor frequency the result of an active suppression operating in vitro? Limiting dilution analyses of CTLprecursor frequencies demonstrate single hit kinetics, implying that if such suppressors exist, they are present in vast excess relative to the numberof target CTLprecursors (23). Removalof donor spleen cells at the time of culture does not relieve the suppression assayed 5 days later (31). Experiments in which spleen cells from suppressed animals were mixed in various ratios with spleen cells from normal animals revealed no suppression of the CTLresponse from the latter spleen cell population (24, 28). Tolerance could not be transferred by spleen cells from suppressed mice injected 14 days previously with minor H different spleen cells (28). The transfer of tolerance by spleen cells from mice injected 4 days previously with a massive dose (108 cells) of minor H different spleen cells probably due to carryover of donor veto cells (8, 26, 32). Thus, all evidence points to the absence of an active mechanismoperating to maintain suppression of CTLprecursors in vitro. The fact that tolerant spleen cells can suppress makesit unlikely there are active suppressors in the spleen cell inoculumother than veto cells. Cells recognizing the idiotype of a CTLreceptor specific for a particular antigen in the context of a given MHCmolecule would be primed by the injection of immunocompetent, receptor-bearing cells (3), and priming has no effect on veto cell frequency(24). If the spleen cell inoculumwere merely responsible for the induction of host suppressor cells, these suppressors would have to have a short lifespan in vivo, would have to be incapable of suppressing in vitro, and wouldhave to be induced passively by antigen and yet distinguish CTLof different restriction specificities. The weakness of the evidence in favor of a suppressor loop and the more compelling arguments against its likelihood have led us to abandon this explanation for in vivo CTLsuppression. One final explanation exists for spleen cell-induced suppression of CTL in vivo that does not involve veto cells. This is the sequestration of these activated CTLand their subsequent exclusion from the cell population taken for the in vitro assay. Several experiments have addressed this possibility, all with negative results. Suppression of the appropriate CTL response is apparent in lymph node cells from normal and splenectomized mice, indicating that the activated precursors are absent from both spleen

Annual Reviews

Annu. Rev. Immunol. 1988.6:115-137. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

126

FINK,

$I~MONKEVITZ & BEVAN

and lymph node, and that the spleen is not required for their specific inactivation (24). Spleen cells inoculated by any of several routes (intravenous, intraperitoneal, subcutaneous, and in the footpad) seem to suppress equally well (24, 28). The activated CTLprecursors are not simply more fragile than naive cells and lost during the process of makinga spleen cell suspension, because diced splenic fragments show the same level of antigen specific suppression as do spleen cell suspensions (24). In summary,the specific CTLsuppression induced in vivo by the injection of antigen-bearing spleen cells bears all the hallmarks of the veto cell-induced suppression previously defined in vitro. It is induced most efficiently by T cells or by cells in the T cell lineage; it is inducedthrough the unidirectional recognition of the inoculated cells by the host and therefore operates directly at the level of the CTLprecursor. It is specific both for antigen and MHC restriction specificity. For all these reasons, we believe that veto cells do operate in vivo to suppress CTLresponses directed at antigens they express, and they can thereby function to help maintain self-tolerance. Do Veto Cells

Kill

the Cells

They Suppress?

The question of whether veto cells suppress primarily by cytolysis is an obvious one. It is raised by the difficulty in reversing suppression and the relative efficiency of suppression mediated by CTLclones and CD8-posifive T cells, relative to helper T cell clones and CD8negative T cells. Previous work suggests that unidirectional recognition leads to unidirectional killing among CTL populations. Thus, when an A anti-B CTL recognizes a B anti-C CTL,the strain B T cell does not lyse the strain A cell (33, 34). More recent work has demonstrated that backwards killing can occur once a T cell receptor is triggered by its appropriate target. In the above example, the strain B CTLclone would lyse the recognizing strain A CTLonly in the presence of strain C target cells (35). The current understanding of the directionality of the killing apparatus makes this result difficult to comprehend(36), although it does fit with our observations that CTLclones are more efficient veto cells whencultured in the presence of monoclonal antibodies directed against their receptors (19). Whatis clear is that CTLclones can serve as veto cells in cultures in the absence of any antigens they specifically recognize (17, 19, 20). These clones are, furthermore, inefficient at lectin-mediated lysis of nontumor cell targets, and this makesit unlikely that veto cells lyse CTLprecursors glued to them by virtue of the CTLreceptor (19). Cloned veto cells able to suppress the generation ofa CTLresponse are unable to kill those same responder cell populations harvested at 17 hr or on day 5 of MLC(19). In other experiments a surprisingly high percentage of cells is apparently

Annual Reviews

Annu. Rev. Immunol. 1988.6:115-137. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

VETO CELLS 127 lysed after 48 hr of MLC (20). It is unknown what this assay was measuring, since this percentageundoubtedlyexceedsthe number of cells specifically respondingto the veto cell alloantigens.Alsoarguingagainst cytolysis as the common meansfor veto cell function is the observation that human helper T cell clones can still proliferate in responseto exogenousgrowth factors at a time whenthey are whollyunresponsiveto antigen (37-39). At least in this system,whichmayor maynot involveveto cells (see below), suppressiondoes not function through respondercell death. In experimentsthat were once considered conclusive evidencc against a requirementfor cytolysis by veto cells, anti-idiotypic antibodies that recognize the T cell receptors of a cloned CTLwere shownto block cytotoxicityby that clone, but not its ability to veto (19). Theseantibodies werecoculturedwith responders, stimulators, and clonedveto cells during the course of a 5-day MLC. Thegeneration of the appropriate CTLactivity was efficiently inhibited, even though antibody-containingsupernatants taken at the end of 5 days completelyblocked cytolysis by the cloned CTL/vetocell both of its specific target and of nonspecifictargets in a lectin mediatedassay (19). Theseresults are equivocal, however,in light of the experimentsdemonstratingbackwardkilling by CTLwhosereceptors are occupied(see above). In other attempts to dissociate CTLactivity fromveto cell function, no correlation wasseen betweenveto activity and cytotoxic activity among CTLclones whoseefficiency of target cell lysis varied with growth(20). Subdonesof CTLthat wereinvariant in cytotoxicity activity wereseen to vary withtheir ability to veto (20). Finally, respondercells fromautologous MLC exhibit veto activity with no demonstrableCTLactivity (18). A11of these data suggest that cytolysis is unlikely to be the major mechanism of veto cell-induced suppression. Is the deletion of anti-veto CTLprecursorsdueto receptor paralysis, a block in the differentiation to effector cell, or someother mechanism? ARE VETO CELLS TOLERANCE?

INVOLVED

IN

ALLOGRAFT

If veto cell-inducedsuppressionis to be acceptedas a meansof maintaining self tolerance, it maybe implicatedin assays morephysiologicallyrelevant than the in vitro assay of CTLfunction. Wenowreinterpret experiments, designed and executed without veto cells in mind, in light of the data alreadypresented.Weadmitthat our selection is not all inclusive, andthat numerousfindings cannot be explained by veto cell-mediated suppressors alone. "This drawbackis, however,hardly peculiar to the veto cell model.

Annual Reviews 128

FINK,

SHIMONKEVITZ & BEVAN

In each of the experimentsdescribedbelow, allograft tolerance is induced in immunodeficienthosts (either neonatal or immunocompromised adult animals)and assayedby the reduction in the graft-versus-host reaction or by the enhancedretention of allogeneic skin or bonemarrowgrafts.

Annu. Rev. Immunol. 1988.6:115-137. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Immunocompromised Adult

Hosts

Adult strain Amice, treated with antilymphocyteserumto removerecirculating T cells, acceptstrain B skin grafted immediately after the injection of strain B bonemarrowcells (40). This enhancedskin graft survival correlated with an antigen-specific reduction in proliferation and CTL activity measured in vitro. Inductionof this in vitro hyporeactivityrequires the presenceof donortype T cells in vivo (40), in obvioussupportof veto cell involvement. In other systemsusing immunodeficient adults, adult strain A miceare given several split doses of lymphoidirradiation followedby an injection of FI(Ax B) bonemarrowcells (41). In these animals, there is long-term survival of strain B skin grafted 1 day after the bonemarrowinjection. Theability of cells differing at minorHantigens to mounta graft-versushost reaction in irradiated hosts is diminishedby prior injection of the donorwith spleen cells bearing the minorHantigens of the host (42, 43). Similarly, as few as 2.5 × 105 cells from a strain ACTLclone bearing irrelevant receptors coinjectedinto irradiated Astrain micewith strain B lymphoid cells protects those recipients fromthe onset of graft-versus-host diseasedueto the B anti-Aresponse(18). Finally, there is evidencefor the prolonged survival of allogeneic and xenogeneicbone marrowchimeras whenirradiated host animalsare coinjected with syngeneicand allogeneic or xenogeneic bone marrowcells (44). This experimental protocol depicted in Figure 4a. In these animals, anti-host activity in the foreign cell inoculummaybe eliminated by veto cells amongthe coinjected host type bonemarrowcells. In each of these cases, lymphoidcells are knownto induce the specific suppressionof immunological activity specific for the antigens of those lymphoidcells, whetherthat activity is measuredas skin or bone marrow graft injection or graft-versus-host reaction. Future work mayreveal whetherthese donor cells are T cells and whether they are required to respondto the host antigens--characteristics that are critical for determiningwhetherthey can be categorizedas veto cells. Neonatal Hosts A large body of workhas demonstratedthat animals can be tolerized to alloantigens by injection, at the time of birth, of lymphoidcells bearing

Annual Reviews VETO

CELLS

129

strainB skin graft 950 red ! 2 weeks

Annu. Rev. Immunol. 1988.6:115-137. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

strain

145 days

acceptance of

A mouse 5x10 strain A + l.SxloTstrainB bone marrow stem cells

strain

B graft

strainC skin graft

acceptanceof strainB graft

4xlo7~ (Axe)

strainB skin graft

bone marrow cells 6-0 weeks

neonatal strain A rat

~ strain C skin yraft

LN, or spleen cells

~0 r~

acceptanceof strain B graft

adult strainA rat strainB skln graft

Figure 4 Veto cell induction of allograft tolerance. (a) Allogeneic radiation bone marrow chimeras showbetter survival and tolerance of strain B skin grafts only upon coinjection of irradiated strain A hosts with bone marrowcells (a source of veto cells) from strains Aand B. (b) The induction of neonatal tolerance, as assayed by the survival of allogeneic skin grafts, maybe mediated by F~ veto cells in the tolerizing bone marrowinocnlum. The transfer of tolerance to irradiated adult animals requires injection of F~cells from the spleen, lymph node, or thoracic duct of tolerant donors.

Annual Reviews

Annu. Rev. Immunol. 1988.6:115-137. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

130

FINK,

SHIMONKEVITZ & BEVAN

those alloantigens (4, 45-50). Thus,strain Arats injected within 24 hr birth with 4 x 107 bone marrowcells from FI(A x B) donors will not reject strain B-skin grafted 6-8 weekslater. This allograft tolerance is radiosensitive (45), antigen specific (third party skin grafted at the same time is rejected), and transferrable. As diagrammed in Figure 4b, as few as 3 x 107 lymphoidcells fromA strain rats tolerized to B alloantigens can transfer the suppressionof B skin graft rejection to irradiated, naive adult Astrain rats (4, 46-48).For our story, the interest lies in whichcells can adoptivelytransfer this tolerance. Neonatallytolerized animalsretain a characteristic lowlevel chimerism in their lymphoid tissues; thoracic duct, bone marrow,lymphnode, spleen, and peripheral blood lymphocytesall harbor an approximatelyuniform2-3%donor type cells. The presence or absenceof chimericF~cells correlates well with the fate of the test skin graft, even in adoptive hosts (47). Removalof chimeric cells from the inoculumwith alloantisera abolishes the transfer of tolerance (47). Toleranceis permanentlyabolished evenin primaryhosts treated in vivo with donor specific antibodies to removechimeric cells (50). These chimeric cells do not simply serve as a source of antigen because thoracic duct lymphocytes transfer tolerance efficiently, while bonemarrowcells do not, although the percentage chimerismin these two populations is identical (47). Evenmoreto the point, elimination of chimeric T but not B cells eliminates the transfer of tolerance (48), while treatment with anti-CD8 plus complementdiminishesbut does not abolish its transfer (4). These results showthat the transfer of neonatally induced tolerance to skin allografts is mediatedby long lived, rapidly recirculating donor type T cells that are radiation sensitive andmainlyCD8positive (45-48). What,if anything, do these tolerogenic donor T cells recognizein the host environment?Accordingto one hypothesis, the cells transferring allograft tolerance bear anti-idiotypic receptors, specifically recognizing all Astrain cells bearinganti-strain B receptors(48). Thereis somedirect evidenceagainst this hypothesis,in experimentsusingthe transfer of allo¯ graft tolerance to the male specific antigen H-Yas a measure for suppression. As in the system described above, both the induction and transfer of tolerance to H-Yin the mouseare mediated by donor T cells and not B cells or macrophages.Toleranceis also conferred, albeit somewhat inefficiently, by an H-Y-bearingCTLclone expressingirrelevant allospecific receptors (49). In this case, no anti-idiotypic interaction possible. Invokingveto cells as the mediatorsof allograft tolerance not only takes into account most of the available data, it is a simplifying hypothesis,alleviating the needfor a specializedcell type andunifyingthe findings fromdiverse systemsmeasuringthe antigen-specific suppression of immunocompetence.

Annual Reviews

VETOCELLS

Annu. Rev. Immunol. 1988.6:115-137. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

ARE VETO CELLS INVOLVED CELL TOLERANCE? "

131

IN T HELPER

In Vivo Experiments Thefirst clues that veto cells mayin somecases suppresshelper T cells were observations madeperipherally in the course of other experiments. Neonatallytolerized rats not only accept allogeneic skin grafts, they make no alloantibodies (47), and they showa diminishedproliferative capacity in response to the appropriate allogeneic stimulators (40, 41, 43, 47). Moredirectly, the proliferative response to lactate dehydrogenaseB in nonresponderstrains of miceis suppressedby the apparent recognition by the helper cell of antigenandI-A on the surface of the suppressoreffector T cell (51). Wewouldlike to suggest (25) that the suppressoreffector cell is a veto cell that bears class II-like moleculeswhichpicks up soluble antigen, and is thereby recognizedby the class II-restrict~d,. antigenspecific helper cell. Thehelper cell is then inactivatedbyits unidirectional recognition of the suppressor cell, succumbingto a veto cell-induced tolerance. Similar meansof suppressionmayoperate in mice that are low respondersto other soluble antigens such as hen egg lysozyme(52). In Vitro Experiments Clonesof humanT cells specific for definedpeptides of influenza hemagglutinin can be tolerized in vitro, in the absenceof other cell types, by their exposureto high levels of antigen for 6-16 hours at 37°C.Tolerance is measuredby the absenceof proliferation in response to immunogenic dosesof peptide pulsedpresentingcells, and it lasts for over 7 days after the removalof antigen and the addition of exogenousgrowthfactors (3739). Toleranceinductionis antigen specific and class II restricted (human T cells expressclass II antigens). Although the mechanism of this tolerance induction is unknown,it is associated with both a loss of the T cell receptor-associated T3 proteins fromthe cell surface and a loss in the ability to bind to monolayersof antigen pulsed presenting cells (39). Importantly,this in vitro-induced tolerance does not result in the death of the suppressedhelper T cells, as their proliferation in the presenceof growthfactors alone is undiminished (37). Thus,T cells specific for class II plus hemagglutininundergoa class II-restricted inactivation in the presence of antigen. At high doses, perhaps enoughantigen binds (either specifically or nonspecifically) to these clonedT cells that someof the peptide associates with class II antigens. This complexcould then be recognizedby neighboringcells that in turn are inactivated. In other words, wethink these experimentsdemonstratethat cloned humanhelper T ceils can serve in vitro as veto cells for class II-restricted responses.Although

Annual Reviews 132

FINK,

SHIMONKEVITZ & BEVAN

Annu. Rev. Immunol. 1988.6:115-137. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

the amountof peptide addedto induce tolerance in this systemsurpasses normalphysiological levels of soluble antigens, wepropose an in vivo modelin whichantigens endogenous to the tolerizing cell are presentedin the context of class II moleculesand serve as the ligand recognizedby the T cell to be suppressed.Clearly, the cells functioningas veto cells for class II-restricted responsesmustthemselvesbear the relevant class II determinants(see below). DO VETO CELLS TOLERANCE?

INDUCE

INTRATHYMIC

Recentexperimentson the ability of fetal thymuslobes to inducetolerance have suggested that bone marrowderived elements in the thymusare much more effective at inducing tolerance to MHC antigens than are thymic e.pithelial or mesenchymal elements(53-55). For example,whenfetal thymuslobes are depleted of macrophages,dendritic cells, and T cell precursors by culturing in deoxyguanosine,the resulting "stromal" lobes retain the ability to attract T cell precursors, to supporttheir maturation, andto select their restriction specificity (56), but they lose the ability inducetolerance to their ownclass I and class II antigens. Thus,a strain A nude mousethat has been grafted with a strain B "stromal" thymus contains T cells whichmaturedin the B thymusbut whichcan respondto B alloantigens. In contrast to this, unmanipulated thymusgrafts do induce tolerance. Thesestudies haveled to the assumptionthat macrophages and dendritic cells in particular are responsiblefor tolerance induction in an untreated thymus. However,these data are compatible with the notion that cells committedto the T lineage also operate in the induction of self-tolerance in a regenerating immunesystem. In recent experimentsaddressing this possibility, Thy-1positive CD4/CD8 negative ("double negative") T stem cells wereisolated fromthe thymusand injected directly into the thymus lobes of lethally irradiated, bone marrow-protectedallogeneic mice. In the situation in whichthe host and T stemcell donormiceexpressedclass I and class II antigen differences, tolerance to the class I antigen was observed, both amongthymocytesand mature T cells, for up to 8 weeks post irradiation and transfer. However,no tolerance wasinducedto donor type class II antigens, whichmacrophages and dendritic cells express but T cells do not. Theselection of Thy-1positive doublenegative thymocytes as the tolerogen and the resulting "split tolerance" suggeststrongly that T cells (and not a minor non-T cell contaminantin the inoculum)are responsible for inducing or maintainingclass I-specific tolerance (57). That is, a subpopulationof immaturethymocytesor their descendantscan

Annual Reviews VETO CELLS

133

serve as veto cells in vivo. Whileit is likely that someof the tolerance is imposed on the regenerating host cells in the thymus by the donor stem cells differentiating simultaneously, it is also conceivable that mature T cells derived from the donor F1 cells recirculate in the periphery and back to the thymus to maintain tolerance (58).

Annu. Rev. Immunol. 1988.6:115-137. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

PERSPECTIVES For the most part, veto cells have been studied in artificial in vitro or in vivo systems in which CTLresponses to alloantigenic differences expressed by the veto cell have been suppressed. Responses against major and minor H differences have been analyzed. This artificial system leads to the proposal that veto cells may be key players in normal immunostasis by inducing and/or maintaining tolerance to self antigens. But what about the potential risks of the existence of a cell that can efficiently suppress an immuneresponse directed against its own antigens? In the systems we have studied, and in particular, in the highly efficient suppression of anti-minor H CTLresponses in vivo (24), the most effective veto cells were CD8positive mature T cells. Analogously, CD8positive CTLclones but not CD4positive helper clones suppressed CTLresponses in vitro (17, 19, 20). If a rather small dose of CD8positive T cells can suppress the generation of a CTLresponse when injected into a host competent to recognize dozens of minor H antigens on the cell surface, then we wouldassume that virally induced antigens expressed by veto cells wouldalso be able to eliminate the anti-viral CTLresponse. For this effect to comeinto play requires that the veto cells becomeselectively infected with the virus. For the effect to be long lasting requires that the immunogenic routes of antigen processing and presentation to CTLare bypassed. Weknow this because in the suppression of the CTLresponse to minor H antigens, the normal route of antigen presentation eventually wins out and the suppression is overcome(24). Whenthe antigenic difference is one that cannotbe effectively presented by the host (i.e. an allogeneic class I difference) then the veto suppression maypersist for a long period (9). If a virus were tropic for T cells and did not damagethe host cell in way that would encourage conventional presentation, T cell and B cell induction wouldbe avoided. The infected T cell, expressing viral antigens on its surface, would function as a veto cell and be immunosuppressive for viral specific T cells, the very lymphocytesimplicated in viral clearance in vivo(59). Another worry is the potential for T cell receptors to becomeloaded with antigen (for exampleclass I molecules complexedwith soluble anti-

Annual Reviews

Annu. Rev. Immunol. 1988.6:115-137. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

134

FINK, SHIMONKEVITZ& BEVAN

gen). Could these exogenous antigens be presented by T cells to other antigen specific T cells as "self" and thereby result in vetoing? To test this alarming possibility, allospecific CTL clones were "loaded" with antigen in vitro and tested for suppression of CTLresponses directed against true endogenous antigens and against these exogenously acquired antigens. Only those responses directed against endogenous antigens were vetoed (17). This argument is undoubtedly a quantitative one, but it does demonstrate that an "average" CTL clone with an "average" number of receptors is unlikely to take up enough antigen, even in vitro, for antigenloaded receptors to serve as ligands for veto cell function. A final limitation of the veto cell model for the maintenance of selftolerance is that it excludes any antigens not expressed by veto cells. Thus in the mouse, where T cells (the most efficient veto cells) do not express class II molecules, class II-restricted responses would not be subject to the same sort of control. In fact, induction of allograft tolerance to class II antigens has been shown to be qualitatively different from that for class I antigens (60~2). Tolerance to these non-T cell expressed antigens may maintained either by inefficient vetoing by other lymphoid and nonlymphoid cells that do express these antigens, or perhaps by some alternative mechanism for deleting autoaggressive cells among the peripheral population. ACKNOWLEDGMENTS This work was supported AI19499 and AI19335.

by US Public

Health

Service

grant

numbers

Literature Cited 1. Good,M. F., Pyke, K. W., Nossal, G. J. V. 1983. Functional clonal deletion of cytotoxic T lymphocyteprecursors in chimeric thymusproducedin vitro from embryonic Anlagen. Proc. NatL Acad. Sci. USA80:3045 2. Kappler, J. W., Roehm,N., Marrack,P. 1987. T cell tolerance by clonal elimination in the thymus.Cell 49:273 3. Bellgrau, D., Wilson,D. B. 1978. Immunological studies of T cell receptors I: Specificallyinducedresistance to graftversus-host disease in rats mediatedby host T cell immunity to alloreactive parental T cells. J. Exp. Med.148:103 4. Roser, B. J., Herbert, J., Godden,U. 1983. The role of suppressor cells in transplantation. Transplant. Proc. 15: 698

5. Cunningham, A. J. 1976. Self tolerance maintained by active suppressor mechanisms. Transplantation31: 23 6. Claesson, M. H., Miller, R. G. 1985. Functionalheterogeneityin allospecific cytotoxic T lymphocyte clones. II: Developmentof syngeneic cytotoxicity in the absenceof specificantigenicstimulation. J. Immunol.134:684 7. Miller, R. G. 1980. Lymphoid suppressor cells. In Strategies of lmmuneRegulation, ed. E. E. Sercarz, A.J. Cunningham, p. 507. NewYork: Academic 8. Rammensee, H. G., Bevan, M. J., Fink, P. J. 1985.Antigenspecific suppression of T cell responses--the veto concept. Immunol. Today 6:41 9. Rammensee, H. G., Fink, P. J., Bevan, M. J. 1985. The veto concept: an eco-

Annual Reviews

Annu. Rev. Immunol. 1988.6:115-137. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

VETO CELLS 135 nomic system for maintaining self tolerance of cytotoxic T lymphocytes. Transplant.Proc. 17:689 10. Miller, R. G. 1986. The veto phenomenon and T cell regulation. Immunol. Today 7:112 11. Miller, R. G., Derry, H. 1979. A cell population in nu/nu spleen can prevent generation of cytotoxic lymphocytesby normalspleencells against self antigens of the nu/nu spleen. J. Immunol.122: 1502 12. Muraoka,S., Miller, R. G. 1980. Cells in bone marrowand in T cell colonies grownfrom bone marrowcan supprcss generation of cytotoxic T lymphocytes directed against their self antigens. J. Exp. Med. 152:54 13. Hurme,M. 1986. Genetic variation in the in vitro veto activity of bonemarrow cells. Scand.J. Immunol.23:389 14. Muraoka,S., Ehrnan,D. L., Miller, R. G. 1984.Irreversibleinactivationof activated cytotoxic T lymphocyteprecursor cells by "anti-self" suppressor cells present in murin¢ bone marrowT cell colonies. Eur. J. Immunol.14:1010 15. Muraoka,S., Miller, R. G. 1983. Cells in murine fetal liver and in lymphoid colonies grownfromfetal liver can suppress generation of cytotoxic T lymphocytesdirectedagainst their self antigens. J. Immunol.131: 45 16. Rammensee, H. G., Nagy,Z. A., Klein, J. 1982. Suppressionof cell mediated lymphocytoxicityagainst minorhistocompatibilityantigens mediatedby LytI + Lyt-2÷ T cells of stimulatorstrain origin. Eur. J. Immunol.12:930 17. Fink, P. J., Rammensee, H. G., Bevan, M.J. 1984.Clonedcytolytic T cells can suppress primary cytotoxic responses directed against them. J. ImmunoL 133: 1775 18. Claesson, M.H, Ropke,C. 1986. Antiself suppressive(veto) activity of respondercells in mixedlymphocytecultures. Curr. Top. MicrobioLlmtnunol. 126:213 19. Fink, P. J., Rammensee, H. G., Benedetto, J. D., Staerz, U. D., I.~francois, L., Bevan,M. J. 1984. Studies on the mechanismof suppression of primary cytotoxic responsesby clonedcytotoxic T lymphocytes. J. ImmunoL133:1769 20. Claesson, M. H., Miller, R. G. 1984. Functionalheterogeneityin allospecific cytotoxic T lymphocyteclones I. CTL clones expressstrong anti-self suppressive activity. J. Exp. Med.160:1702 21. Miller, R. G. 1980. An immunological suppressorcell inactivating cytotoxic T lymphocyteprecursor cells recognizing

it. Nature287:544 22. Miller, R. G., Phillips, R. A. 1976. Reductionof the in vitro cytotoxic lymphocyte response produced by in vivo exposureto semiallogeneic cells: r~ruitmentor active suppression?J. Immunol. 117:1913 23. Rammensee, H. G., Fink, P. J., Bevan, M.J. 1984. Functional clonal deletion of class I-specific cytotoxic T lymphocytesby veto calls that expressantigen. J. lmmunoL133:2390 24. Fink, P. J., Weissman, I. L., Bevan,M. J. 1983. Haplotypespecific suppression of cytotoxic T cell inductionby antigen inappropriatelypresentedon T cells. J. Exp. Med. 157:141 H. G., Juretic, A., Nagy, 25. Rammensee, Z. A., Klein, J. 1984.ClassI restricted interaction between suppressor and cytolytic cells in the responseto minor histocompatibilityantigens. J. lmmunol. 132:668 26. Gascoigne,N. R. J., Crispe, I. N. 1984. Suppression of the cytotoxic T cell responseto minoralloantigens in vivo: Linked recognition by suppressor T cells. Eur. J. Immunol.14:2t0 27. Ishikawa, H., Suzuki, H., Hino, T., Kubota,E., Saito, K. 1985.In vivo priming of mouseCTLpr~ursors directed to product of a newlydefined minor H-42 locus is undera novelcontrol of class II MHC gene. J. Immunol.135:3681 28. Ishikawa, H., Hino, T., Kato, H., Suzuki, H., Saito, K. I986. Cytotoxic T lymphocyteresponse to minor H-42a alloantigen in H-42bmice: clonal inactivation of the precursor cytotoxic T lymphoeytes by veto like spleencells that express the H-42aantigen. J. Immunol. 137:2080 29. Bevan, M. J. 1976. Minor H antigens introducedon H-2different stimulating cells cross react at the cytotoxicT cell level duringin vivopriming.J. Irnmunol. 117:2233 30. Shearer, G. M., Polisson, R. P. 1980. Mutualrecognition of parental and F~ lymphocytes. Selective abrogation of cytotoxic potential ofF~ lymphocytes by parental lymphocytes.J. Exp. Med.151: 20 31. Rammensee, H. G., Bevan, M. J. 1987. Mutualtolerization of histoineompatible lymphocytes.Eur. J. Immunol.17: 893 32. Crispe, I. N., Owens,T. 1985. Veto in vivo? Immunol.Today 6:40 33. Kuppers,R. C., Henney,C. S. 1976.Evidencefor direct linkage betweenantigen recognitionandlytic expressionin effector T cells. J. Exp. Med.143:684

Annual Reviews

Annu. Rev. Immunol. 1988.6:115-137. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

136

FINK, SHIMONKEVITZ& BEVAN

34. Kuppers, R, C., Henney,C. S. 1977. Studies on the mechanism of lymphocyte mediatedcytolysis. IX. Relationships betweenantigen recognition and lytic expressionin killer T cells. J. Immunol. 118:71 35. Lanzavecchia, A. 1986. Is the T cell receptor involved in T cell killing? Nature 319:778 36. Kupfer, A., Swain, S. L., Janeway,C. A., Singer,S. J. 1986.Thespecificdirect interaction of helper T cells and antigen presentingBcells. Proe.NatLAead.Sci. USA 83:6080 37. Lamb,J. R., Skidmore, B. J., Green, N., Chiller, J. M., Feldmann,M. 1983. Inductionof tolerancein influenzavirusimmuneT lymphocyteclones with synthetic peptides of influenza hemagglutinin. J. Exp. Med.157:1434 38. Lamb,J. R., Feldmann,M. 1984. Essential requirement for major histocompatibility complexrecognition in T cell tolerance induction. Nature308:72 39. Feldmann,M., Zanders, E. D., Lamb, J. R. 1985. Tolerancein T cell clones. Immunol. Today 6:58 40. Maki, T., Gottschalk, R., Wood,M. L., Monaco,A. P. 1981. Specific unresponsiveness to skin allografts in anti-lymphocyteserum-treated, marrow injected mice: participation of donor marrow-derivedsuppressor T cells. J. lrnmunoL127:1433 41. Slavin, S., Strober,S., Fuks,Z., Kaplan, H. S. 1977.Inductionof specific tissue transplantation tolerance using fractionated total lymphoidirradiation in adult mice: long-termsurvival of allogeneic bonemarrowand skin grafts. J. Exp. Med. 146:34 42. Halle-Pannenko,O., Pritchard, L. L., Rappaport, H. 1983. Alloimmunization-activatedsuppressorcelts. I. Abrogation of lethal graft-versus-hostreaction directed against non-H-2antigens. Transplantation36:60 43. Pritchard, L. L., Halle-Pannenko, O. 1983. Alloimmunization-activatedsuppressorcells. II. In vitro activity of suppressor cells implicated in the abrogation of lethal graft-versus-host reaction. Transplantation36:310 44. Ildstad, $. T., Sachs,D. H. 1984.Reconstitution with syngeneieplus allogeneic or xenogeneic bone marrowleads to specific acceptanceof allografts or xenografts. Nature307:168 45. Holan, V., Hasek,M., Chutna, J. 1978. Radiosensitivity of suppressorcells in neonatallytolerant rats. Transplantation 25:27 46. Roser, B., Dorsch,S. 1979.Thecellular

basis of transplantationtolerancein the rat. Immunol.Rev. 46:55 47. Dorsch, S., Roser, B. 1982. Suppressor cells in transplantation tolerance. I. Analysisof the suppressorstatus of neonatally and adoptively tolerized rats. Transplantation33:518 48. Dorsch,S., Roser, B. 1982.II. Identification and probable modeof action of chimeric suppressor T cells. Transplantation 33:525 49. Weissman,I. L., Jerabek, L., Greenspan, S. 1984. Tolerance and the H-¥ antigen: requirementfor male T cells, but not B cells, to inducetolerance in neonatal female mice. Transplantation 37:3 50. Lubaroff, D. M., Silvers, W.K. 1970. Theabolition of tolerance of skin homografts in rats withisoantiserum.J. bnmunoL 104:1236 51. Baxevanis,C. N., Ishii, N., Nagy,Z. A., Klein, J. 1982. H-2-controlled suppression of T cell response to lactate dehydrogenaseB. Characterization of the lactate dehydrogenaseB suppressor pathway. J. Exp. Med.156:822 52. Araneo, B. A., Yowell, R. L. 1985. MHC-linked immune response suppression mediatedby T cells bearing IA-encodeddeterminants. J. Immunol. 135:73 53. von Boehmer, H., Hafen, K. 1986. Minorbut not major histocompatibility antigens of the thymusepithelium tolerize precursors of cytolytic T cells. Nature 320:626 54. Jenkinson,E. J., Jhittay, R., Kingston, R., Owen,J. J. T; 1985. Studies on the role of the thymieenvironmentin the induction of tolerance to MHC antigens. Transplantation39:331 55. Schuurman,H. J., Vaessen, L. M. B., Vos,J. G., Hertogh,A., Geertzema,J. G. N., Brandt, C. J. W.M., Rozing, J. 1986. Implantation of cultured thymic fragmentsin congenitally athymicnude rats: ignorance of thymic epithelium haplotype in generation of alloreactivity. J. ImmunoL 137:2440 56. Lo, D., Sprent,J. 1986.Identity of cells that imprint H-2restricted T cell specificity in the thymus.Nature319:672 57. Shimonkevitz,R. P., Bevan,M.J. 1987. Tolerogenie interactions amongthymocytes. Manuscriptsubmitted 58. Fink, P. J., Bevan,M. J., Weissman, I. L. 1984. Thymic cytotoxic T lymphocytes are primed in vivo to minor histocompatibility antigens. J. Exp. Med. 159:436 59. Brenan,M.1983. T lymphocytesin viral clearance, lmmunoLToday 4:319

Annual Reviews VETO CELLS

137

Annu. Rev. Immunol. 1988.6:115-137. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

comparisonof the neonatal tolerance60. Streilein, J. W., Gruchalla,R. S. 1981. Analysisofneonatallyinducedtolerance inducing capacities of H-2class I and of H-2alloantigens. I. Adoptivetransclass II antigens. J. Immunol.131:1670 fer indicates that tolerance of class I 62. McCarthy,S. A., Bach, F. H. 1983. The and class II antigens is maintainedby cellular mechanismof maintenanceof distinct mechanisms.Immunogenetics neonatally induced tolerance to H-2 12:161 class I antigens. J. Immunol.131:1676 61. McCarthy,S. A., Bach, F. H. 1983. A

Annu. Rev. Immunol. 1988.6:115-137. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Annu. Rev. Immunol. 1988.6:115-137. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Annual Reviews www.annualreviews.org/aronline Ann. Rev. lmmunol. 1988. 6:139-59 Copyright © 1988 by Annual Reviews lnc. All rights reserved

Annu. Rev. Immunol. 1988.6:139-159. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

ANTIGENIC VARIATION IN LENTIVIRAL DISEASES Janice E. Clements,’~ Susan L. Gdovin,~f Ronald C. Montelaro* and Opendra Narayan~f ~" The Johns HopkinsUniversity School of Medicine, Departmentof Neurology,600 N. WolfeStreet, Baltimore, Maryland21205, and * Departmentof Biochemistry,Louisiana State University and Louisiana Agricultural ExperimentStation, Baton Rouge,Louisiana 70803 INTRODUCTION Virus infections in animals involve complexvirus-host interactions in whichvarious schemesof replication of the agent are pitted against a multitude of host-defense mechanisms.The immunesystem is the most effective of the antiviral host defensesandusuallysucceedsin eliminating pathogensafter brief periods of infection. Failure to cure infection indicates a shift in favor of the virus, and this involves either somenovel mechanism of viral replication that eludes the immune systemor a defect in the immune system, often virus induced, that interferes with antiviral functions. This review examinesone modeof viral persistence in which normalreplication of the agent can occur in the functionally competent immuneenvironment.The basic mechanism of this process is mutation of the virus genomewith attendant alteration in surface antigens permitting escape from immunecontrol. Thelentiviruses are a uniquegroupof agentsthat elude the host defense systemsand that not only persist indefinitely in the infected animalbut replicate continuously, often in the face of competentimmune responses (1-3). Theseagents are nononcogenic retroviruses that infect cells of the immunesystem. Theyare host species-specific and are transmitted horizontally fromindividual to individual in bodyfluids. Theviruses belong to a taxonomicgroup that includes visna-maedivirus of sheep, caprine arthritis encephalitis virus (CAEV) of goats, equineinfectious anemiavirus 139 0732-0582/88/0410-0139502.00

Annual Reviews 140

CLEMENTS ET AL

(EIAV) of horses, human immunodeficiency virus in humans (HIV), simian immunodeficiency virus in non-humanprimates (SIV) (4, 5, Within this group of viral infections, host responses to visna-maedi, CAEV and EIA viruses have been the most thoroughly investigated, and in this report we summarize various strategies used by these agents to escape immunological control.

Annu. Rev. Immunol. 1988.6:139-159. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

ANTIGENIC

DRIFT

AND PERSISTENCE

Antigenic drift or change in the antigenicity of the surface glycoprotein is a well-established mechanism among parasites for escaping immune elimination (7). It is the principal meansof persistence of protozoans such as trypanosomes,plasmodia, and several species of bacteria. Recent studies suggest that this phenomenonmay also be important in persistence of several viruses. The best-known exampleof viral antigenic drift is among influenza viruses where variants with minor antigenic changes in the hemagglutinin and/or the neuraminidase glycoproteins are selected during sequential epidemics by populations immuneto preceding strains of virus (8). Recent studies showthat variant strains of rabies and measles virus can replicate in the presence of antibody to other strains of the samevirus, suggesting antigenic variation (9, 10). The best modelfor viral drift in single animal, however,comesfrom studies of infections with lentiviruses. The primary examples are equine infectious anemia (EIA) (l 1-13) ovine visna (14, 15). Horses infected with equine infectious anemia virus develop sequential episodes of acute hemolytic crises during persistent infection. The animals produce neutralizing antibody to the virus after the first episode, but succeeding disease crises are associated with mutant viruses that are not neutralized by the preexisting antibody. This is reminiscent of protozoan diseases. Visna virus of sheep also causes persistent infection, although episodic disease does not occur. However, the agent undergoesantigenic drift in the animal during persistent infection in a way similar to EIA in horses. Such antigenic drift by visna virus could be duplicated in cell culture systems using plaque-purified virus for inoculation and monospecific antibody for selection (15, 16). These viruses provide ideal modelsfor studying the viral genetic mechanismsof antigenic variation. In addition to antigenic drift the caprine virus, CAEV,escapes from immuneelimination by molecular masking of the viral epitopes responsible for neutralization. This permits the virus to persist in an immunocompetent animal. However, when neutralizing antibodies are produced experimentally, analysis of virus stocks with these antibodies reveals an extremely high rate of antigenic variation.

Annual Reviews

ANTIGENS IN LENTIVIRAL DISEASES 141

Annu. Rev. Immunol. 1988.6:139-159. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

LENTIVIRUSES Lentiviruses are a subgroupof the retrovirus family. Their viral genome is a positive stranded RNAmolecule(9500 bases) whichcontains three genes that encodethe major structural proteins of the virus 07). The env gene encodes the viral glycoproteins (external and transmembrane proteins) whichcomprisethe envelopecoat of the virus (17). Thegag gene encodesthe viral core proteins and a viral RNA binding protein. The pol gene codes for a complexof enzymesinvolvedin reverse transcription of the viral RNAinto proviral DNA and integration of the proviral DNA; these include the RNA-dependent DNA polymerase(reverse transcriptase, RNaseH, DNA endonuclease-integrase). The genes are organized on the RNA genome(5’-3’)gag-pol-env. Lentiviruses have a genetic organization similar to the other retroviruses with the addition of small open reading frames (ORF)betweenthe po! and env genes (17, 18). These ORFsprobably code for proteins that play roles in the comp}exregulation of gene expressionof these viruses. The envelope gene of visna virus and CAEV encodesan outer membrane protein of 135 kd whichbecomeshighly glycosylated and whichis the target for virus neutralizing antibodies (19, 20). The outer membrane protein of EIAVis 90 kd, highlyglycosylated,and the target for antibodies that neutralize the virus (21-23). All three viruses have transmembrane proteins whichare glycosylated and hydrophobic, and whichapparently span the membrane of the virus serving as an anchor for the outer membrane protein. The outer membrane proteins of the lentiviruses are substantially larger than other retrovirus envelopeglycoproteinsand appear to be more highly glycosylated. These common features maycontribute to their ability to accumulate mutationsyet maintaina functional structure on the surfaceof the virus. Anotheractivity of the outer membrane glycoprotein is to induce cell fusion. Bothvisna virus and CAEV causeextensive fusion of infected cells, andvisna virus can causecell fusion fromoutsidethe cell at multiplicities of infection of four virions per cell (23, 24). This fusion by visna virus can be blocked by monospecificantibody to gp135, the outer membrane glycoprotein. Thus, the outer membrane glycoproteins of the lentivirus contain epitopes responsible for virus neutralization, and probablyfor virus-cell receptor interaction andvirus-inducedcell fusion. Thelife cycle of lentiviruses providesan obviousmechanism for eluding host immunedefenses: Viral RNAis transcribed by RTaseinto proviral DNAwhichintegrates into the host cell genomeand can be eliminated only by the deathof the cell (26). However, since the target cells for these viruses are end stage cells of the monocyte-macrophage lineage (27, 28),

Annual Reviews

Annu. Rev. Immunol. 1988.6:139-159. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

142

CLEMENTS ET AL

integration of the proviral DNA provides only temporarysurvival of the viral genome.The virus must replicate and disseminate to other target cells in order to persist for the life of the animal.Since virions budding fromcells are potentially accessibleto neutralizingantibodies,lentiviruses have evolved multiple strategies to evade the humoralimmunityof the immunocompetent host. First, epitopes responsiblefor virus neutralization are not recognizedby the immune systemof the host. This results in the failure of the host to produceneutralizing antibodies while it producesa plethora of non-neutralizingantibody against the gp135as well as other viral proteins. Thelack of neutralizingantibodiesallowsthe virus to persist andreplicate in the animal.Second,neutralizing antibodies are inducedin infected animalsby someviruses. However, these antibodies are inefficient in virus neutralization, requiring a prolongedincubation with virus to accomplish efficient neutralization, and are of relatively low titer compared to neutralizing antibodies directed against other viruses. Virus can spread fromcell to cell beforeneutralization occurs; againthis results in a persistent infection. Third, lentiviruses sustain frequent point mutations throughouttheir genomeduring replication; this is due to an error-prone viral RTasewhichlacks editing functions. Point mutationsin the env gene of a variant virus that alters the neutralization epitope provide a strong selective advantage for growth in an immuneanimal. The resulting nonneutralized virus can replicate and spread in the immunehost. Thus, antigenic variation provides a mechanism for the virus to persist in the immune host. TheCAEviruses use the first two strategies while visna and EIAviruses use the secondand third. IMMUNE RESPONSE

TO CAEV

Goats infected naturally or experimentallywith CAEV becomepersistently infected andproduceantibodiesthat bind to all the viral proteins (29, 30). However, these antibodiesare ineffective in neutralizing the virus (29-31). Thelack of neutralizingantibodiesin infected goatsis difficult to overcome; even experiments in which adjuvants were used together with purified and disrupted virus did not succeedin eliciting a neutralizing antibody response. However,recent studies havebeensuccessful in eliciting a neutralizing antibody response to CAEV whenanimals are inoculated with virus in conjunction with large amountsof inactivated Mycobacterium tuberculosis (30). Studies on the neutralizing antibody haveshownthat they are of extremelynarrowspecificity. The serumneutralizes only the virus used for inoculation, no strains of visna virus or other strains of CAEV are recognized. This includes CAEviruses isolated from goats from the sameherd from whichthis neutralized virus had been derived. This

Annual Reviews

Annu. Rev. Immunol. 1988.6:139-159. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

ANTIGENS IN LENTIVIRAL DISEASES 143 suggests these viruses are not only poor antigens for the induction of neutralizing antibodiesbut that various strains bear distinct neutralization epitopes. Since CAEV does not normallyinduce neutralizing antibodies, it is not possibleto determinewhetherdifferent antigenic types of virus are present in different animalsor whetherthere are antigenicdifferencesin the viruses obtained from the sameanimal. Thus, the neutralizing sera obtained by injection of inactivated M. tuberculosis and virus were used to determine if antigenically distinct viruses werepresent in stocks of CAEV. It was foundthat 1 in 1 × 104parental virus wasnot neutralizedby the antibodies (32, 33). This is an extremelyhigh rate of spontaneousantigenic mutation since these variants arose in stocks withoutthe selective pressure of neutralizing antibody.Theoccurrenceof unselectedantigenic variants in visna virus stocks is at least 100 times lower(34). This high rate of mutation mayprovide an explanationfor the antigenic variability of CAEV in nature without the obviousselective pressure of neutralizing antibodies. Further evidenceof this high variability comesfrommolecularanalysis of three cloned strains of CAEV, all of whichhave widely divergent restriction endonucleasemaps(20, 35-38). PERSISTENT INFECTION AND ANTIGENIC VARIATION OF VISNA VIRUS Sheepinfected naturally or experimentallywith visna virus becomepersistently infected with the virus despite the productionof neutralizing antibodies3-4 weeksafter infection(14, 39-41 ). Visnavirus canbe isolated fromthe circulating blood monocytesof these animalsat all times after infection (41-43). Early studies on persistent infection in sheephad shown that virus isolated from the animals late in the infection could not be neutralized by serumantibodies (44). These results suggested that the presenceof neutralizing antibodyis not sufficient to clear the virus infection, that virus mayundergomutation during persistent infection, and that virus variants no longer neutralized by the antibodies mayhave selective growthadvantage. Twoaspects of this antigenic variation seen in persistent infection should be emphasized:(a) Variant viruses do not appearto replace the infecting strain of virus, and(b) antigenicvariation of visna virus in sheepcan only be detected in 25 %of animals.Tworecent studies concludedfromthese observationsthat antigenic variation is rare and thus could account neither for viral persistence nor for continued replication of the virus in immune animals(45, 46). However,that variant viruses do not replace the infecting strain should not be surprising since these viruses are retroviruses that integrate into the host genome.In

Annual Reviews 144

CLEMENTS ET AL

Annu. Rev. Immunol. 1988.6:139-159. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

addition, studies of persistently infected sheep showthat monocyteprecursors in the bonemarrowcontain viral nucleic acid providinga reservoir of the infecting strain (as well as other strains) in stemcells in the bone marrow(43). That with pooledpolyclonalsera antigenic variation is found in one quarter of all infected animalssuggests that this is a biologically significant phenomenon. In addition, monoclonalantibodies can detect subtle antigenic changesin "parental" type virus that are not detected by pooledserum(47). MOLECULAR STUDIES OF VISNA VIRUS

OF ANTIGENIC

VARIANTS

Todeterminethe molecularbasis of antigenic variation westudied antigenicvariants of visna virus fromtwoinfected sheepas well as visna virus mutants selected in culture in the presence of early immunesera. Two sheep (1 and 4) were inoculated with plaque-purified virus. At various intervals during a 3-year period, blood from the animals providedserum for measuringneutralizing antibody,and white bloodcells that werecocultivated with sheep fibroblasts for virus isolation (15, 16). Theearliest antibodies producedby the sheep neutralized the parental virus (strain 1514)used for inoculation but riot viruses isolated later frombloodleukocytes (sheep viruses LV1and LV4). The antibody responses broadened with time, and sera obtainedlate in the infection neutralized the variant viruses (Table 1). However,the last serumsamplefrom each sheep could still distinguish the parental andvariant viruses, emphasizing the antigenic difference of the agents. To determinewhether the variant viruses may havepreexistedin the virus suspensionused for inoculation of the animal, weexamineda concentratedpreparation of plaque-purifiedvisna virus for the presence of variants. Astock of parental virus containing 1 7x 10 plaque-formingunits (pfu) was mixedwith early immunesera and examined for non-neutralizablevariants. After 6 hr of incubationat 37°C,followedby overnightincubationat 4°C, all survivingviruses hadthe parental phenotype,i.e. less than 1 infectious virus in 1 x 107 of parental virus was antigenicallydistinct (33, 34). In contrast to CAEV, therefore, visna virus has a low rate of spontaneousantigenic mutation. The variant viruses in the animal must therefore have developed under selective pressure by antibodiesduringthe persistent infection. To test the ability of immune sera fromthese animalsto select variants in tissue culture, weinoculatedcell cultures with 103pfu of plaque-purified virus 1514 and maintained the cultures with mediumcontaining early immunesera from the two animals (16, 47). Viruses developingin these dultures (tissue culture variants AD1-1 and AD4-1)were no longer neu-

Annual Reviews ANTIGENS IN LENTIVIRAL DISEASES

145

Table 1 Genetic changes in antigenic variants ofvisna virus strain 1514, isolated from two persistently infected sheep and afrom tissue culture

Annu. Rev. Immunol. 1988.6:139-159. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Virus

Neutralization Early Late

Genetic changes

1514

++++

++++

LV1-2 LVI-7 LV 1-4 L¥ 1-3 LV1-6 LV1-5 LV1 - 1

++++ -------

++++ + + + + + + + + +

AB AB AB ABC ABC ABC ABCDEF

LV4-2 LV4-3 LV4-1

+ + + + ---

+ + + + + + ++

A EF A EF A C EF

ADI-1 AD4-1

---

÷ ÷ + +

AB AB

E E

a TheLVIviruses wereisolated fromsheep1 and the LV4viruses fromsheep 4. The tissue culture derived virus variants ADI-Iand AD4-1were selected with early immunesera from sheep 1 and 4, respectively.Thegeneticchangesare thoselocatedin the 3’ regionof the genome.

tralized by the sera used to select them and were thus antigenic variants similar to those arising in the animal. These in vitro-derived viruses were comparedwith those variants obtained from the animals during persistent infection (Table 1). Viral antigenic drift has been postulated to result from point mutations in the viral genome, which produce changes in the outer membraneglycoprotein, gp135. As a first step in investigating the molecular basis for the altered antigenicity of the naturally occurring variants, as well as of the tissue culture-derived mutants, the genomic RNAsof these viruses were compared by RNaseTl-resistant oligonucleotide fingerprinting (34, 48). Eighty unique RNAseTl-resistant oligonucleotides were identified, and their locations were mappedon the viral RNA.Sequence analysis was performed (a) to determine whether the unique oligonucleotides in the different viruses were the same, and (b) to determine the differences between altered oligonucleotides. These experiments showedthat the antigenic variants were closely related to the parental virus and differed from it by only a few changes localized in the env region of the viral genome.

Annual Reviews

Annu. Rev. Immunol. 1988.6:139-159. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

146

CLEMENTS ET AL

Further analysis showedthat manyof the changes were commonamong the variants, reflecting the samepoint mutation. This suggestedthat the changesaccumulatedin the genomesof the variant viruses and that antigenic drift occurred in a progressive manner(Table 1). Oligonucleotide changesoutside the env gene were rare, and no one changewas observed in morethan one virus. This suggested that these mutations were random and had no selective advantage for the virus carrying them. However, neutralizing antibody in the immuneanimal appeared to have a strong selection for mutationalchangesin the env geneof visna virus, and these changeswere maintainedand passed to progenyvirus. Theprogressivenature of antigenic mutationof visna virus in sheep is illustrated by the changesdetected in the viruses isolated fromsheep 1. Virus LV1-2,isolated from this sheep, wasof parental serotype; however, it was found to differ from the parental virus by two oligonucleotides located in the env gene (Table 1 summarizesthe genetic changes and serologic characterization of the variants from two sheep). This change wasfoundin all subsequentantigenic variants fromthis animal, suggesting that all the variants descendedfromthis virus and that it had a selective advantageover the parental virus. Theoriginal serological classification of the variants (Table1) is consistent with the cumulativechangesobserved in the env gene. Whenvariants from a second infected animal (sheep 4) were examined, it was surprising to find that the same oligonucleotide changes were detected and that these changes reflected the same point mutations, as determinedby sequence analysis. These common changes in the variants fromthe two animalssuggestthat the selective pressure for the phenotypic changein the viral antigenicity requires very specific alterations in the viral genome,since mutations probably occurred randomly.The selected mutationspresumablyspecifically alter the site of interaction of the neutralizing antibodymoleculewith the viral glycoprotein. This mightinvolve three or four epitopes on the protein, each containing a small numberof aminoacids. Further support for this highly specific selection of particular point mutationscomesfrom RNaseTi fingerprint analysis of antigenic variants isolated in tissue culture. Early immune sera fromsheep 1 and 4 wasused to select variants in tissue culture, AD1-1 and AD4-1.Theoligonucleotide changesdetected in these viruses werea subset of the changesfoundin the antigenic variants isolated fromthe persistently infected sheep(Table1). Thus,the samepoint mutationalchangeswereselected in vitro and altered the antigenic phenotypeof these tissue culture-derivedvariants. To determinethe exact number,position, and potential biological role of particular mutationsin the antigenic variants of visna virus, clones of

Annual Reviews

Annu. Rev. Immunol. 1988.6:139-159. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

ANTIGENS IN LENTIVIRAL DISEASES

147

a numberof the variants from sheep 1 have been obtained, and the completenucleotide sequenceof the enveloperegion has been determined. In LVI-1, the most distant variant, the envelopegene contains only 11 nucleotide changes from the parent strain 1514. Previous RNaseT~ oligonucleotidemapping,whichonly resolves 10-15%of the genetic information, detected 6 changes. Therefore a larger numberof changes were expected. Despite the dramatic difference in the antigenic phenotypeof LVI-1fromthe parental phenotype,it differs fromthe parental virus by only a small numberof point mutations. Further only 7 of the 11 base changesresulted in changesin aminoacids in the envelopeproteins, and only six of these aminoacids are in the outer membrane protein. At the carboxyl terminus of the outer membrane protein, there is a cluster of three aminoacid changes, and two of these represent changes from unchargedaminoacids in the parental strain to chargedaminoacids in the variant LVI-I. Thus, this site mayrepresent a region of the outer membrane protein that is important in recognition by the immuneresponse. It is interesting that noneof the aminoacid changesaltered potential glycosylationsites in the envelopeproteins. Thus,relatively few changes in the amino acid composition of the gp135 appear to have profound effects on the antigeniccharacterof the protein. Studies using monoclonalantibodies against the gp135of visna virus strain 1514further demonstratedthat small numbersof base changesin the variants hadprofoundeffects on the topological arrangement of epitopes in this protein (47). Thesestudies suggested that point mutations in the env gene result in the exposureof epitopes previously present in the glycoprotein, rather than creation of newantigenic sites. Themonoclonal antibodies prepared against gp135 defined five partially overlapping epitopes which comprise two domains. One domainis called an overt domainbecause it is exposedon the native virus; the other domainis covert and does not appear to be accessible to antibodyon native virus. All five epitopes defined by the monoclonal antibodies werealtered in the antigenic variants from sheep 1. Nosimilar changeswere detected in the core protein p27 whensimilar analyses were done with anti-p27 monoclonal antibodies. Binding of the highest avidity MAbsto epitopes gp135a, gp135b, and gp 135c, whichare exposedon virus strain 1514,decreasedduringantigenic drift; these three epitopeswereobscuredor lost on the variants (Figure1). However,whenthe LV1viruses were treated with nonionic detergents or reducing agents, they then regained MAb-binding abilities against these three epitopes. This suggests that these surface epitopes weresometimes blockedor buried rather than lost during antigenic variation. Bindingof lower-avidity MAbsto epitopes gp135d and gp135c, whichare covert or

Annual Reviews 148

CLEMENTS

ET

AL

1.6 1.2 0.8 0.4

G8

Annu. Rev. Immunol. 1988.6:139-159. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

0.6 0,4 0.2

~J O~ 0,6

02

0.2

0.8

0,8

0.6

0.6

0,4

0.4

0.2

0.2 i~ 9P 1§14 LV LV LV LV LV LV 135 I-2 I-4 i-3 I-5 I-6 I-I

Qp 1514 LV LV LV LV LV LV 135 I-2 I-4 I-3 I-5 I-6 I-I Figurel

Topographical

MAb binding

The LV1 viruses divergence

are

from strain

against

epitop~s

epitope

might

density

changes

to purified

(b)

in visna

gp 135 during

gp 135 (strain ordered

along

1514), the

abscissa

1514.

The five

gp135 epitopes

and (e)

displayed

two distinct

be subdivided

at 492 nm; S.D.,

(subscripts

standard

antigenic

strain

1 and

1514, from

left

drift,

or the to

right

are marked (a) ELISA patterns, 2).

shown by differential

six LV1 antigenic

Abbreviations:

by increasing through

(e)

genetic

(boxes);

which suggested O.D.

variants.

that

492 nm, optical

deviation.

less accessible on strain 1514, increased during antigenic drift; these two epitopes were reciprocally exposed or unblocked on the variants (Figure 1). Treatment of strain 1514 with reducing agents increased MAbbinding to covert epitope gp135e three- to five-fold. This suggests that disulfide bonding on gp135 may diminish MAbbinding to epitope gp135~ on strain 1514; enhanced MAbbinding to this epitope on the LV1 viruses may be

Annual Reviews

Annu. Rev. Immunol. 1988.6:139-159. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

ANTIGENS IN LENTIVIRAL DISEASES

149

due to conformational rearrangements of gp135 which involve altered disulfide linkages. MAbsagainst epitope gpl35~1 bound the LV1viruses better than they boundstrain 1514, just as they had boundgpl35 purified from strain 1514better than they boundthe virus itself. This suggests that during antigenic variation, the covert gp135~1 epitope may have been exposed on the LV1viruses in a conformation resembling that caused by detergent treatment and purification of gp 135 from the envelope of strain 1514. By ELI SA, MAbsagainst epitopes gp135e through gp 135d clearly differentiated strain 1514 from its closest progeny, LV1-2, whereas the antibodies in early- or late-post infectious immunesheep sera had been unable to distinguish the two viruses in neutralization tests. Paradoxically, by ELISA, the MAbsdid not separate LV1-2 from LVI-1, LV1-3, or LV14, whereasserum neutralization tests had separated them. It is of interest that the anti-gp 135 MAbswe used did not neutralize visna virus. Thusthe five epitopes on gp135 recognized by our MAbsmay be distinct from the epitopes responsible for neutralization. This suggests that antigenic drift is associated with complex rearrangements of both neutralizing and nonneutralizing epitopes on the visna virus envelope glycoprotein. These data suggest that topographical flip-flop or refolding of the tertiary structure of gp135 may phenotypically amplify genetic changes in variable regions of the env gene. Topographical flip-flop of gp135 might further explain the symmetrical losses and gains of MAb-bindingsites on the viral glycoprotein during antigenic drift. Three epitopes exposed on strain 1514 (gp135a, gp135b, gp135c) were lost or obscured on the LV1 viruses, while two covert epitopes on strain 1514(gp 135~ and gp 135¢) were reciprocally exposed or unblocked on the LV1mutants. Topographical permutability may dramatically increase the amount of information stored on the envelope glycoprotein and mayallow visna virus to sequester biologically active epitopes from the immunesystem. Wehad earlier observed that neutralizing antiserum from a sheep infected with visna virus strain 1514 did not neutralize the mutant LV1-1virus that was isolated later from the same sheep (15). However,whenanother sheep was hyperimmunizedwith nonionic detergent-disrupted strain 1514, the animal developed antibodies that neutralized both strain 1514 and the LVI-1 virus. This suggests that strain 1514 contains the neutralization epitope(s) of LV1-1, but that this site on its envelopeglycoprotein is conformationally concealed or blocked until it is exposedto the immunesystem by detergent solubilization. Such exposure of conformationally hidden neutralization epitopes on strain 1514 gp135 strikingly resembles the manner in which covert epitopes gp135d and gp135~ were topographically exposed or unblocked on the LV1viruses during antigenic drift in this study. Cor-

Annual Reviews 150

CLEMENTS ET AL

Annu. Rev. Immunol. 1988.6:139-159. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

relation of the genetic analysis of the antigenic variants with the monoclonal antibody studies suggests that a small numberof changes in the aminoacid compositionof visna virus gp135has a profoundeffect on the structure of the virus envelope. The mechanismby whichthese changes alter the structure appears to be changingthe epitopes exposedon the surface rather than directly changingan epitopeor creating a newantigenic site. CLINICAL COURSE AND IMMUNE RESPONSES DURING PERSISTENT EIAV INFECTION Theclinical responsesof horses followingnatural or experimentalinfection by EIAVcan be dividedinto characteristic stages (1, 50). AcuteEIA(fever and hemorrhages)is typically associated with the first exposureto virus andappears to correlate with massivevirus replication in and destruction of macrophages. In the initial stages of EIAthe animalsare seronegative, but maybecomeseropositive, developing neutralizing antibody, within 16-30 days post infection. The earliest and predominant antibody responsesare directed against the EIAVenvelopeglycoproteins, gp90and gp45. Minorantibody responses can then be detected against the major internal proteins of the virus, whichare 10-20times moreabundantin the virus than the viral glycoproteins. The moreclassical symptoms of EIA(anemia, weight loss, edema,etc) are observedlater during recurring cycles of viremia and illness which appearat irregular intervals separated by weeksor months.This stage of disease is called chronic EIA.Horses with chronic EIAare seropositive, and their T-lymphocytesare responsive to purified, inactivated EIAV. There is no evidence of depression of humoral or cellular immune responses. However, the magnitude of humoral immuneresponses to EIAVcan vary markedly during the course of chronic EIA, and cellfree EIAVis frequently found in the blood as infectious antibody-virion complexes(51). Thefrequencyand severity of clinical episodes decline with time, and the chronic stage of disease usually ends within the first year after infection, after an averageof 6-8 disease episodes. Duringthis stage of disease the virus can be efficiently transmitted by wholeblood transfers from infected to naive horses via insect vectors (52) or humans (hypodermic needles, scalpels, etc). At this point, the infected horse enters the inapparent stage of EIA during whichthere are no clinical symptomsor detectable viremia. The horse continues to harbor EIAV,however,as evidenced by the fact that disease can be efficiently transmittedby experimentalwholebloodtransfers to naive horses. In addition, it has beendemonstratedthat recrudescence

Annual Reviews

Annu. Rev. Immunol. 1988.6:139-159. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

ANTIGENS IN LENTIVIRAL

DISEASES

151

of chronic EIAcan be induced in someinapparently infected horses by administration of dexamethasone or by stress, even in animals that have beenfree of febrile episodesfor years (53). Thus, the periodic nature of EIAoffers a uniquely dynamicmodelfor examininglentivirus persistence in the presence of fully competenthost immuneresponses. Moreover,EIAV-infectedhorses represent a system in whichthe infected animal routinely establishes control of lentivirus replication, presumablyresulting from cumulativeimmune responses generated during the first year post infection. Clarifying the immune status by whichthe horse eventuallycontrols EIAVreplication, evenin the face of relatively rapid antigenicvariations of the virus, shouldprovidevaluable insights into strategies for the immunologicmanagement of lentivirus infections. NATURE OF ANTIGENIC VARIATION PERSISTENT EIAV INFECTION

DURING

The uniqueperiodic nature of chronic EIAcan evidently be attributed to the sequential evolution of antigenic variants of EIAVthat temporarily escape established humoraland cellular immuneresponses. The initial evidence for EIAVantigenic variation is based on comparative neutralization assays employingvirus isolates and immunesera recovered fromchronically infected horses duringvarious stages of disease (54, 55). Morerecently a detailed characterization of EIAVantigenic variation has been madepossible by the developmentof an animal model system in whichShetland ponies inoculated with a standard strain of EIAVreproducibly develop chronic EIA, during which time EIAVisolates can be efficiently recoveredin the sequential cycles of disease (11). Apanel over 20 of these virus isolates has been characterized by a variety of immunologic and biochemicalproceduresto ascertain the nature of antigenic variation in EIAV(12, 56-59). Themajor conclusions of this survey can be demonstratedby examining the properties of a panel of 13 virus isolates recovered during febrile episodes in three experimentallyinfected ponies (Table 2). Eachvirus isolate wasinitially examined in neutralization assays usingserumsamples collected during sequential afebrile periods in the infected ponies. The results of these assays reveal several importantproperties of persistent EIAVinfections. First, within each group of virus isolates froma single pony, each isolate can only be inactivated by serumsamplestaken after the clinical episodeof origin; noneof the virus isolates can be neutralized by serumsamplestaken prior to the disease cycle of origin. Second,crossneutralization assays indicate that each isolate is antigenically distinct,

Annual Reviews 152

CLEMENTS ET AL Table 2 Characterization of EIAVisolates obtained during sequentialclinicalepisodesin threeponiesexperiencing chronicEIA inducedbyexperimental infection witha standardvirus inoculum (11). Theglycoprotein epitopeswereidentifiedin eachvirusisolated basedon reactivity in immunoblots with a panel of monocional antibodies(60)

Annu. Rev. Immunol. 1988.6:139-159. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

EIAV Isolates Pony91:P3.1-1 P3.1-2 P3.1-3 P3.1-4 Pony127:P3.2-1 P3.2-2 P3.2-3 P3.2-4 P3.2-5 Pony135:P3.3-1 P3.3-2 P3.3-3 P3.3-4

Isolationday post inoculation 12 32 58 198 13 41 99 137 158 16 46 75 107

Epitopes gp90 gp45 ABCD ABCDE ABF AE AD ABCD ABCDF AE AE ACD ABCDF ABCDF A

A A A A AB AB AB AB AB A AB AB AB

although an occasional low level of cross reactivity can be detected in the neutralization assays. The antigenic uniqueness of most isolates can also be demonstrated by assaying the reactivity in Western blots using a panel of monoclonal antibodies specific for gp90 and gp45, respectively. The panel of 16 gp90-specific monoclonalantibodies define four distinct, but overlapping, epitopes (designated A, B/F, C/D, and E), two of which (C/D and E) are involved in virus neutralization. The panel of eight gp45-specific monoclonal antibodies defines two distinct, non-overlapping epitopes (designated A and B), neither of which is involved in virus neutralization. Thus, these antigenic analyses indicate that a distinct antigenic variant is associated with each febrile episode in an experimentally infected animal and that the evolution of a new predominant strain of virus can take as little as two weeks or as long as several months. There is no detectable common pattern of variant evolution in the parallel infections in different ponies. Thus, EIAVantigenic variation differs from that observed in visna virus in that the former is evidently more rapid and morerandom(i.e. less selected upon). The biochemical nature of antigenic variation amongthe panel of EIAV isolates has been examined both by peptide and glycopeptide mapping procedures to compare viral proteins and by oligonucleotide fingerprint

Annual Reviews

Annu. Rev. Immunol. 1988.6:139-159. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

ANTIGENS IN LENTIVIRAL DISEASES

153

analysis to comparegenomicRNA(12, 56-59). The peptide/glycopeptide variations are specific for the viral envelopeglycoproteins(gpg0and gp45); no structural alterations are observedamongthe internal viral proteins of the respective virus isolates. Thecombinationof peptide and glycopeptide mappingindicates that each of the 13 virus isolates contain structurally unique gp90 and gp45 components, as no two maps were identical in pairwise comparisons.Moreover,analysis of the variant peptides detected amongsequential virus isolates fromeach ponyreveals that peptide alterations appearingin oneisolate are not necessarily retained in the next or successivevariants in that pony.Similarly,the oligonucleotidefingerprints differentiate amongall of the virus isolates and onceagain demonstratea noncumulativepattern of variation amongsequential isolates. Pairwise comparisons of oligonucleotidefingerprints typically identify 3-4 variant oligonucleotidesout of a total of over 50 oligonucleotidesresolved for each genomicRNA.The degree of protein and genomicvariation detected by these proceduressuggests that the antigenic variation observedin EIAV is the result of randompoint mutations in the viral RNA,someof which result in altered glycoproteinaminoacid sequences.Alterations in peptide mapsand oligonucleotide fingerprints are not detected during passage of the virus isolates in tissue culture, indicating the requirementof immune selection in generating newpredominantvariants in horses. In these aspects, EIAVantigenic variation closely resembles variation in visna virus. However,the apparently noncumulative and completely random nature of EIAVvariation is in markedcontrast to the cumulative,common patterns of visna virus variation describedabove. To relate the observedantigenic and structural variations directly to nucleotide sequencevariation in EIAV,the env gene sequences of four virus isolates obtained from pony91 weredeterminedand compared(59). All but one difference in nucleotide sequenceamongthe four isolates resulted fromsingle base substitutions; a single three-base insertion was observedin isolate P3.2-5. Figure 2 summarizesthe deducedaminoacid sequencefor the gp90and gp45components of each virus isolate. Overall, the nucleotide divergencebetweenany pair of virus isolates ranges from 16-37 bases out of a total of 2583bases in the env gene. Approximately 75%of all nucleotidesubstitutions result in aminoacid replacements,and aminoacid divergencerangesfrom11-29residues out of 860 total. In light of the extent of variation observedin peptide mapping,the relatively low level of nucleotide (0.6-1.4%) and aminoacid (1.3-3.4%) divergence somewhatsurprising. However,the majority of all amino acid substitutions are nonconservative,andlikely to induce changesin structure and in antigenic properties. Variation is not evenly distributed throughout the eno gene. Twoto

Annual Reviews 154

CLEMENTS ET AL

leader

< > gp90

Annu. Rev. Immunol. 1988.6:139-159. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

P3.2-1 HVSI AFYGG l pGGI STP I TQOSEKSKCEEHTMFQPYCYI~NSHAE~KEARDQEH#LKEESKEEKRANDMA(I GHFLLCLAGTTGG I L~EGL~QH t t ~~ t P3.2-2 ~ E P3.2-3 P3.2-5 I ~

P3,2-I I AQFGKCILWSH I GH~ l F~ILGAS I I KY I VMFLLI YLLL]TSSPK I LRALWI(VTSGAGSSGGRYLKKKFHHKHASREDTkl)QAQHH I HLAGVTGGSGDKYYKQ C P3.2-2 S Y t P3.2-3 ~ S N C P3.2-5 S Y t P3,2"1 LF KYSRND~NGE$EEYN~PKS~KS~EAF~ES~SEKTKGE]SQPGAA[NEHK~GGNNPH~G~LDLE~EGGN~Y~CC~KAr~EGTLA~PCCGFPLW ~~ tt tt 1" ~_ R P3.2-2 P3.2-3 ~ P3.2-5 T ~ P3.2-IWGLVIIVGRIAGYGLRGLAV[ I R ] CI RGLNLI FE I I RKHLDYI GRALNPGTSHVSMPQYV P3.2-~ P3.Z-3 P3.2-5 F

Figure 2 Deducedamino acid sequences for the env genes of EIAVisolates from pony 91. The complete sequence is shownfor isolate P3.2-1 while amino acid acids begins with the initiator methionine. Regions corresponding to the leader peptide, the highly glycosylated outer membraneglycoprotein, gp90, and the proposed transmembrane glycoprotein, gp45, are indicated. Cysteine residues are indicated by arrows (T T). Potential N-linked glycosylation sites are highlighted by shaded boxes. Within gp90, conserved regions are indicated by lines above the aminoacid sequence, and the hypervariable region is indicated by asterisks (*). The proposed precursor cleavage site (RHKR)is designated by an arrow (,~). Putative membranespanning regions with gp45 are indicated with square brackets ([ ]).

Annual Reviews

Annu. Rev. Immunol. 1988.6:139-159. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

ANTIGENSIN LENTIVIRALDISEASES

155

three times as manyamino acid substitutions occur in the exterior gp90 as do in the transmembrane gp45. Further, within gp90, two conserved and one hypervariable region can be identified. The first conserved region within gp90 includes the N-terminal 21 amino acid leader peptide and the following 1 l0 aminoacids, while the second conserved region encompasses aminoacid residues 370 to 445. In these regions, there are few substitutions, and all potential N-linked glycosylation sites and cysteine residues are absolutely conserved amongthe four isolates. In contrast, a variable region of gp90 spans amino acid residues 143 and 366, in which pairs of virus isolates differ by 3% to 9% in amino acid sequences. A hypervariable region of 35 amino acids (residues 306-338) is located within the gp90 variable region. Within this hypervariable region, amino acid divergence between any two isolates ranges from 3%to 20%. EIAVgp90 is highly glycosylated and contains numerouspotential N-linked glycosylation sites (N-X-S/T) within the variable region of the protein. Since about 40% all aminoacid substitutions in the env gene involve the loss or gain of an asparagine (N) residue, it is perhaps not surprising that the number glycosylation sites within the variable region of gp90range from six to ten in the different virus isolates. In contrast to gp90, EIAVgp45 is more highly conserved among the four virus isolates, with amino acid substitutions between isolate pairs ranging from 0.7% to 2% (3-9/415 residues). Within gp45, the amino terminal half is more highly conserved than the carboxyl terminal half of the molecule, including two hydrophobic (putative transmembrane) regions (residues 448-473 and 617-636) and four N-linked glycosylation sites that are conserved amongall isolates. The variant sequence data described above indicate that the antigenic differences amongthe virus isolates examinedcan be correlated with relatively minor differences in aminoacid sequence, most ofwhiohare localized to a relatively small region of gp90. It is not yet possible to correlate epitopes defined by monoclonalantibodies with specific peptide sequences. However,it is interesting to note that limited proteolytic digestions of EIAVgp90 produce fragments of about 12,000 tool wt which react with all of the neutralizing and most of the non-neutralizing monoclonalantibodies (Montelaro, unpublished). Thus, it is tempting to speculate that the antigenic sites maybe localized in the variable/hypervariable region of gp90. The sequence data also permit estimation of the mutation rate of the EIAVenv gene during persistent infection. This calculation uses the formula R = D/2T (61), where R is the number of nucleotide substitutions per site per year, D is the divergence calculated from the proportion of nucleotide differences between pairs of isolates, and T is the divergence time represented here as the time betweenthe isolation of the first isolate

Annual Reviews 156

CLEM~NT$ET AL

(P3.2-1) and the later isolates. Usingthese parameters,mutationrates 1 × 10-1, 2 × 10-2, and 1 × 10-2 were obtained for isolates P3.2-2, P3.2-3, and P3.2-5, respectively. Thesemutation rates are about 10-fold higher than those observedfor HIV(62) and 1000-foldgreater than observedfor other RNA viruses (63).

Annu. Rev. Immunol. 1988.6:139-159. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

CONCLUSIONS Antigenicvariation of the lentiviruses as exemplifiedby visna virus and EIAVposes a formidable problem to the host immunesystem as well as to those designingvaccinesagainst these agents. It is clear fromthe studies described in this review that immunizationwith the undenaturedenvelope protein of any onelentivirus will not protect against the rapidly emerging variants that occur during infection with these viruses. The studies describedhere suggestthat there are hypervariableregions in the envelope genes of these viruses. The molecular mechanismof this high rate of variability in a small genetic region mayprovide clues for genetically engineering novel immunogenswhich would provide a broad range of neutralizingantibodiesto lentiviruses. Indeedmutationappears to be only one aspect of the complexantigenic phenotypeof the envelopeproteins of lentivirus, the three dimensional structure of the outer membraneprotein also determines recognition of epitopes by the host immunesystem. Thus, studies on the structure and organizationof the envelopeproteins will be importantfor the design of effective vaccinesagainstlentiviruses. ACKNOWLEDGMENTS

This workwassupportedby grants fromthe National Institutes of Health NS21916, NS16145, NS07000and CA38851. Wewould like to thank Linda Kelly for preparation of the manuscript. Literature Cited I. Cheevers,W.P., McGuire,T. C. 1985. cytopathic retroviruses (HTLV-III)from Equineinfectious anemiavirus. Immupatients with AIDSand pre-AIDS.Scinopathogenesis and persistence. Rev. ence 224:497-500 4. Gonda,M. A., Wong-Staal,F., Gallo, Infect. Dis. 7:83-88 2. Narayan, O., Cork, L. C. 1985. LenR. C., Clements, J. E., Narayan, O., tiviral diseases of sheep and goats. Gilden, R. V. 1985. Sequencehomology Chronic pneumonia,leukoencephalitis and morphologicsimilarity of HTLV-III and visnavirus, a pathogeniclentivirus. and arthritis. Rev. Infect. Dis. 7: 8998 Science 227:173-77 5. Gonda,M. A., Braun, M. J., Clements, 3. Popovic, M., Sarngadharan, M. G., Read,E., Gallo, R. C. 1984.Detection, J. E., Pyper, J. M., Casey,J. W.,Wongisolation and continuousproduction of Staal, F., Gallo, R. C., Gilden, R. V.

Annual Reviews

Annu. Rev. Immunol. 1988.6:139-159. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

ANTIGENSIN LENTIVIRALDISEASES 1986. HTLV-IIIshares sequence homology with a family of pathogeniclentiviruses. Proc.Natl. Acad.Sci. USA83: 4007-11 6. Murphey-Corb, M., Martin, L., Rangan, S., Baskin, G., Gormus, B., Wolf, R., Andes, A., West, M., Montelaro, R. 1986. Isolation of an HTLV-III-related retrovirus from macaqueswith simian AIDSand its possible origin in asymptomaticmangabeys.Nature 321: 435-37 7. Bloom,B. R. 1979. Gamesparasites play: howparasites evade immune surveillance. Nature279:21-26 8. Webster, R. G., Laver, W.G., Air, G. M., Schild, G. C. 1982. Molecularmechanismsof variation in influenza virus. Nature 296:115-21 9. Wiktor, T. J., Koprowski,H. 1980. Antigenic variants of rabies virus. J. Exp. Med. 152:99-112 10. Ter Muelen,V., Loftier, S., Carter, M. J., Stepbenson,J. R. 1981. Antigenic characterization of measles and SSPE virus haemaglutinin by monoclonal antibodies. J. Gen.Virol. 57:357~4 11. Kono,Y., Kobayashi,K., Fukunaga,Y. 1973.Antigenicdrift of equineinfectious anemia virus in chronically infected horses. Arch. Virusforchung41:1-10 12. Orrego,A., Issel, C. J., Montelaro,R. C., Adams,W.V. 1982. Virulence and in vitro growthof a cell-adaptedstrain of equineinfectious anemiavirus after serial passagein ponies.Am.J. Vet. Res. 43:1556-60 13. Montelaro,R. C., Parekh, B., Orrego, A., Issel, C. J. 1984.Antigenicvariation during persistent infection by equine infectious anemiavirus, a retrovirus. J. Biol. Chem.259:10539-44 14. Narayan,O., Griffin, D. E., Chase,J. 1977.Antigenicshift of visna virus in persistently infectedsheep.Science197: 376-78 15. Narayan,O., Griffin, D. E., Clements, J. E. 1978.Virus mutationduring "slow infection." Temporaldevelopmentand characterization of mutants of visna virus recovered from sheep. J. Gen. Virol. 41:343-52 16. Narayan,O., Clements,J. E., Griffin, D. E., Wolinsky,J. S. 1981. Neutralizing antibody spectrumdeterminesthe antigenic profiles of emergingmutants of visna virus. Infect. Immunol.32: 104550 17. Sonigo, P., Alizon, M., Staskus, K., Klatzman, D., Cole, S., Danos, O., Retzel, E., Tiollais, P., Haase,A., WainHobson,S. 1985. Nucleotide sequence of the visna lentivirus: Relationshipto

157

the AIDSvirus. Cell 42:369-82 18. Davis,J. L., Molineaux, S., Clements,J. E. 1987. Visnavirus exhibits a complex transcriptional pattern: Oneaspect of geneexpressionshared withthe acquired immunodeficiency syndromeretrovirus. J. Virol. 61:1325-31 19. Vigne,R., Filippi, P., Querat,G., Sauze, N., Vitu, C., Russo,P., DeLori,P. 1982. Precursor polypeptides to structural proteins of visna virus. J. Virol. 42: 1046-56 20. Pyper, J. M., Clements, J. E., Molineaux, S. M., Narayan,O. 1984. Genetic variation amo.n~%lentiviruse~: Homologybetween vlsna virus a~d capfine arthritis-encephalitis virus is confined to the 5’ gag-polregionanda small portion of the env gene. J. Virol. 51: 713-21 21. Montelaro,R. C., Lohrey, N., Parekh, B., Blakeney,E. W., Issel, C. J. 1982. Isolation and comparativebiochemical properties of the major, internal polypeptides of equine infectious anemia virus. J. Virol. 42:1029-38 22. Montelaro,R. C., West, M., Issel, C. J. 1983.Isolation of equine infectious anemiavirus glycoproteins.Lectinaffinity chromatography proceduresfor high avidity glycoproteins. J. Virol. Methods 6:337-46 23. Montelaro,R. C., West,M., Issel, C. J. 1984. Antigenicreactivity of the major glycoproteinof equineinfectious anemia virus, a retrovirus. Virology136:368-74 24. Harter, D. H., Choppin,P. W.1967.Cell fusing activity of visna virus particles. Virology31: 279 25. Narayan,O., Clements,J. E., Strandberg, J. D., Cork, L. C., Griffin, D. E. 1980. Biologiccharacterization of the virus causingleukoencephalitisarthritis in goats. J. Gen.Virol. 50:69-79 26. Haase, A. T., Varmus,H. E. 1987. Demonstration ofa DNA provirusin the lytic growthof visna. NatureNewBiol. 245: 237-39 27. Narayan,O., Wolinsky,J. S., Clements, J. E., Strandberg,J. D., Griffin, D. E., Cork,L. C. 1982.Slowvirus replication: the role of macrophagesin the persistence and expressionof visna viruses of sheep and goats. J. Gen. Virol. 59: 345-56 28. Narayan, O., Kennedy-Stoskopf, S., Sheffer, D., Griffin, D. E., Clements,J. E. 1983.Activationof caprinearthritisencephalitis virus expression during maturation of monocytes to macrophages. Infect. Immun.41:67-73 29. Crawford, T. B., Adams,D. S. 1981. Caprinearthritis encephalitis: clinical

Annual Reviews

Annu. Rev. Immunol. 1988.6:139-159. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

158

CLEMENTSET AL

features and presence of antibody in selected goat populations. J. Am.Vet. Med. Assoc. 178:713-19 30. Narayan,O., Sheffer,D., Griffin, D. E., Clemems,J. E., Hess, J. 1984. Lackof neutralizingantibodiesto caprinearthritis-encephalitislentivirus in persistently infected goats can be overcome by immunizationwith inactivated Mycobacteriumtuberculosis.J. ViroL49: 34955 31. Klevjer-Anderson,P., McGuire,T. C. 1982. Neutralizingantibodyresponse of rabbits and goats to caprine arthritisencephalitis virus. Infect. lmrnun.38: 45541 32. Narayan,O., Clements,J. E., KennedyStoskopf, S., Royal,W.1986. Antigenic variation in ovinecaprine lentiviruses. In AntigenicVariationin Infectious Diseases, ed. T. H. Birkbeck,C. W.Penn, pp. 25-40. Oxford:IRL Press 33. Narayan,O., Clements,J. E., KennedyStoskopf, S., Royal, W. 1987. Mechanismsof escape of visna lentiviruses from immunologicalcontrol. In Antigenic Variation: Molecularand Genetic Mechanisms of RelapsingDisease, ed. J. M. Cruze, pp. 60-76. Basel: S. Karger Publ. In press 34. Clements, J. E., D’Antonio, N., Narayan, O. 1982. Genomicchanges associated with antigenic variation of visna virus. II. Common nucleotide changesdetected in variants fromindependent isolations. J. Mol. BioL 158: 415-34 35. Roberson, S. M., McGuire, T. C., Klevjer-Anderson, P., Gorham,J. R., Cheevers,W.P. 1982. Caprinearthritisencephalitisvirus is distinct fromvisna and progressive pneumoniaviruses as measured by genome sequence homology. J. Virol. 44:755-58 36. Chiu,I. M., Yaniv,A., Dahlberg,J. E., Gazit, A., Skuntz,S. F., Tronick,S. R., Aaronson, S. A. 1985. Nucleotide sequenceevidence for relationship of AIDSretrovirus to lentiviruses. Nature 317:366-68 37. Pyper, J. M., Clements, J. E., Gonda, M. A., Narayan, O. 1986. Sequence homology between cloned CAEVand visna virus,58:665-70 twoneurotropiclentiviruses. J. Virol. 38. Yaniv,A., Dahlberg,J. E., Tronick,S. R., Chiu, I. M., Aaronson,S. A. 1985. Molecularcloning of integrated caprine arthritis encephalitisvirus. Virolo#y145: 340-45 39. Narayan,O., Griffin, D. E., Silverstein, A. M. 1977. Slowvirus infection: Replication and mechanism of persistence of

visnavirus in sheep.J. Infect. Dis. 135: 800-6 40. Griffin, D. E., Narayan,O., Adams,R. J. 1978.Early immune responsein visna, a slowviral diseaseof sheep.J. Infect. Dis. 138:340-50 41. Narayan,O., Wolinsky,J. S., Clements, J. E., Strandberg,J. D., Griffin, D. E., Cork,L. C. 1982.Slowvirus replication: The role of macrophagesin the persistence andexpressionof visnavirus of sheepand goats. J. Gen.Virol. 59: 34556 42. Gendelman,H. E., Narayan, O., Kennedy-Stoskopf,S., Kennedy,P. G. E., Ghotbi, Z., Clements,J. E., Stanley, J., Pezeshkpour, G. 1986. Tropismof sheep lentiviruses for monocytes:Susceptibility to infection and virus gene expression increase during maturation of monocytesto macrophages.J. ViroL 58:67-74 43. Gendelman,H. E., Narayan, O., Molineaux, S., Clements,J. E., Ghotbi, Z. 1985.Slowpersistent replication of lentiviruses: Role of tissue macrophages and macrophage-precursors in bone marrow.Proc. Natl. Acad. Sci. USA82: 7086-90 44. Gudnadottir, M. 1974. Visna-maediin sheep. Prog. Med.Virol. 18:336-49 45. Thormar, H., Barshatzky, M. R., Kozlowski,P. B. 1983. The emergence of antigenicvariation is a rare event in long term visna64:1427-32 virus infection in vivo. J. Gen. Virol. 46. Lutley,R., Petursson,G., Palsson,P. A., Georgsson,G., Klein, J., Nathanson,N. 1983.Antigenicdrift in visna: virus variation duringlong term infection of Icelandic sheep. J. Gen. Virol. 64: 143340 47. Stanley, J., Bhaduri, L. M., Narayan, O., Clements,J. E. 1987. Topographical rearrangementsof visna virus envelope glycoproteinduring antigenic drift. J. Virol. 61:1019-28 48. Dubois-Dalcq, M., Narayan, O., Griffin, D. E. 1979.Cell surfacechanges associated with mutationof visna virus in antibody treated cell cultures. VirolotTy 92:353~56 49. Clements, J. E., Petersen, F. S., Narayan, O., Haseltine, W. S. 1980. Genomicchanges associated with antigenicvariation of visna virus duringpersistent infection. Proc.Natl. Acad.Sci. USA77:4454-58 50. Issel, C., Coggins,L. 1979.Equineinfectious anemia: Current knowledge. J. Am. Vet. Med.Assoc. 174:727-33 51. McGuire,T., Crawford,T., Henson,J. 1972.EIA:Detectionof infectious virus-

Annual Reviews

Annu. Rev. Immunol. 1988.6:139-159. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

ANTIGENSIN LENTIVIRALDISEASES

159

tious anemiavirus during parallel perantibody complexesin the serum. Immusistent infections. J. Virol. 61:1266-70 nol. Commun. 1:545-51 52. Issel, C., Foil, L. 1984.Studieson EIAV 59. Payne, S., Fang, F. D., Lui, C. P., transmission by insects. J. Am. Vet. Dhruva,B., Rwambo, P., Issel, C., Montelaro, R. 1987.Antigenicvariation and Assoc. 184:293-97 lentivirus persistence: variations in 53. Kono,Y., Hirasawa,K., Fukunaga,Y., Taniguchi, T. 1976. Recrudescenceof envelope gene sequences during EIAV EIA by treatment with immunosupinfection resemblechangesreported for pressive drugs. Natl. Inst. Anim.Health sequential isolates of HIV.Virology,In press Q. 16:8-15 54. Kono,Y., Kobayaski,K., Fukunaga,Y. 60. Hussain,K., Issel, C., Schnorr,K., West, 1973.Antigenicdrift of equineinfectious M., Rwambo,P., Montelaro, R. 1987. anemiavirus in chronically infected Epitope mappingof the envelope proteins of EIAV. J. Virol. In press horses. Arch. Virol. 41:1-10 S. 1985. Rates 55. Kono,Y., Kobayaski,K., Fukunaga,Y. 61. Gojobori,T., Yokoyama, 1971. Serological comparisons among of evolutionof the retroviral oncogene strains of EIAV.Arch. Virol. 34:202-8 of Moloneymurine sarcomavirus and 56. Payne,S., Parekh,B., Montelaro,R. C., of its cellular homologues.Proc. Nail Acad. Sci. USA82:4198-4201 Issel, C. J. 1984. Genomic alterations associated with persistent infections by 62. Hahn, B., Shaw, G., Taylor, M., equine infectious anemiavirus. J. Gen. Redfield, R., Markham, P., Salahuddin, ViroL 65:1395-99 S., Wong-Staal, F., Gallo,R., Parks, E., Salinovich, O., Payne, S., Montelaro, 57. Parks, W.1986. Genetic variation in HTLV-III/LAV over time in patients R., Hussain,K., Issel, C., Schnorr,K. 1986. Rapid emergenceof novel antiwith AIDSor risk for AIDS.Science genic and genetic variations of equine 232:1548-53 nfectiousanemiavirus duringpersistent 63. Holland,J., Spindler,F., Horodyski,E., infection. J. Virol. 57:71-80 Grabau,E., Nichol, S., VanDePol, S. 58. Payne, S., Salinovich, O., Nauman, S., 1982. Rapid evolution of RNA genomes.Science 215:1577-85 Issel, C., Montelaro,R. 1987. Course andextent of variation of equineinfec-

~

Annu. Rev. Immunol. 1988.6:139-159. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Annu. Rev. Immunol. 1988.6:139-159. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Annual Reviews www.annualreviews.org/aronline

Annu. Rev. Immunol. 1988.6:161-195. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Ann. Rev. lmmunol. 1988. 6 ." 161-95 Copyright © 1988 by Annual Reviews Inc. All rights reserved

STRUCTURE, ORGANIZATION, AND REGULATION OF THE COMPLEMENT GENES R. Duncan Campbell, S. K. Alex Law, Kenneth B. M. Reid, and Robert B. Sire Medical Research Council ImmunochemistryUnit, Department of Biochemistry, University of Oxford, South Parks Road, OxfordOX13QU, United Kingdom

INTRODUCTION cDNA and genomicclones are nowavailable for nearly 30 glycoproteins whichcomprisethe components,regulatory proteins, and receptors associated with the complement system(Figure 1, Table1). Thesecloningstudies have provided information which emphasizes the mannerin which the membersof the complementsystem maybe divided into families of structurally andfunctionally related proteins manyof whichare codedfor on the samechromosome and are closely linked. This review is divided into six mainsections in order to illustrate these structural andgenetic relationships: (a) the Clqr2szcomplexand Cl-inhibitor; (b) C2, factor C4, and21-hydroxylase,the class III genesof the majorhistocompatibility complex;(c) C5and the thioester containing proteins C3and C4; (d) regulators of complementactivation, (the RCAlinkage group) on human chromosome1 which includes the plasma proteins factor H and C4bbinding protein (C4bp)and the membrane molecules complementreceptor type 1 (CR1),complement receptor type 2 (CR2),decay accelerating factor (DAF)and probably membranecofactor protein (MCP);(e) complement receptor type 3 (CR3) as a memberof the cell adhesion glycoprotein family; and (f) the componentsof the terminal attack complexC5b-C9. 161 0732-0582/88/0410-0161502.00

Annual Reviews 162

CAMPBELL,

LAW, REID

& SIM

Annu. Rev. Immunol. 1988.6:161-195. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

ACTIVATION AND CONTROL OF THE COMPLEMENT SYSTEM Themainfeatures of activation andcontrol of the systemare outlined below. Moreextensive background informationis given in other recent reviews(1, 2, 3). Thereare twodistinct routesof complement activation, the classical andalternative pathways(Figure1), with the majorcomponentC3(1.3 mg/ml)playinga central role in each. TheC3and12 other plasmaglycoproteinsformthe 13 components of the system(Figure 1). Controlis mediatedpartly by 7 plasmaproteins andpartly by various (about15) membrane boundproteins andreceptors; Table1. Theclassical pathwayis consideredto be triggered primarily by the interaction of subcomponent Clq of the C1complexwith the Fc regions oflgG, or IgM, in immunecomplexes.Alternative pathwayactivation can be mediated by a widerangeof nonimmunoglobulin activators such as fungi, yeast, lipopolysaccharides,etc, althoughcomplexesof IgG, IgA, andIgE may also cause activation. Theearly activation steps, or controlof activated components, involve the splitting of only one or twopeptidebondsby one

ENZYMECOMPLEXES WHICHACTIVATEC3 ANDC5

EARLYACTINGCOMPONENTS

TERMINAL COMPONENTS

CLASSICAL / PATHWAY - - - -~J ACTIVATOR ~ C 4-~--~

C 4b .~.

C 4bC 2--~---~

C4b2a

C3 C3a

ca(H~oI;’~ ALTERNATIVE J~ PATHWAY ~J ACTIVATOR - - Yl C3(H20),~,~)

~

~i

_

~ Surface C3b+ Site +CS--]J---~ - CSb~C5b-9 ~C3a

/

Ba

¯ Denotes proenzyme form ° Denotes activatedform

Figure 1 An outline of the activation of the classical and alternative pathways is shown. The system is down-regulated, as outlined in Table 1, mainly by: Cl-Inh (for activated Clr and Cls); C4bp and factor I (for C4b); factors H and I (for C3b); anaphylatoxin inactivator (for C3a, C4a, C5a); S-protein (for C5b-9); CR1 and factor ] (for C3b and C4b); DAF(for C3b and C4b); MCPand factor I (for C3b). Properdin, unlike the other control proteins has an enhancing effect on activation via its stabilization of the C3bBbcomplex.

Annual Reviews

Annu. Rev. Immunol. 1988.6:161-195. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

COMPLEMENTSYSTEM GENES

163

of six highly specific serine proteases (Figure 1) or by a carboxypeptidase B-like enzyme(Table 1). In classical pathwayactivation, the Clq (460 kd) molecule,whichhas no enzymicactivity, interacts via its six globular headgroupswith aggregated Fc regions of immunecomplexes.This binding appears to allow a conformational change to take place within the Clq Clr2Cls2 complex allowing autoactivation of the proenzymeform of Clr (83 kd) which turn activates proenzyme Cls (83 kd). Thethree chainC4molecule(~, 95 /3, 70kd; y, 33 kd) is split byCis at a single point in its 0¢ chainto yield the anaphylatoxin C4a(9 kd) and the large C4bfragment. The freshly activated C4bhas the capacity to bind covalently to hydroxylor amino groups via a reactive acyl group of a glutamyl residue derived from a thioester bondin its ~’ chain. This feature, whichis shared with C3b, allows these large activation fragmentsof C4and C3to bind to a variety of surfaces by ester or amidebonds and is probably of someimportance in the generation of C3 and C5 convertases and also in clearance mechanisms. Thethird serine protease of the classical pathway,proenzyme C2 (102 kd), associates (probably via its N-terminal C2bdomain)with in a Mg2+-dependent fashion andthen is split by Cis to yield a noncatalytic chain C2b(30 kd) and a catalytic chain C2a(70 kd). Theresulting C4b2a complexis the C3convertase of the classical pathway,splitting C3(~, 115kd;/3, 75 kd) at onepoint in its 0¢ chainto yield the anaphylatoxin C3a (9 kd) and the large fragmentC3b. There are two serine proteases in the alternative pathway,proenzyme factor B (90 kd) and factor D (24 kd) (whichhas only beenfound in activated formin plasma). In activation of the alternative pathway,factor B associates (via its N-terminal Ba portion) with C3b, or C3(H20) C3b-likeformof C3), in a Mg2 ÷_dependent interaction whichallowsfactor Dto split B into its N-terminal,noncatalytic, Ba chain (30 kd) andits terminal, catalytic, Bb chain (60 kd), thus yielding the C3convertase C3bBb.Efficient activators of the alternative pathwayare consideredto possesssites for C3bwhereit is protectedfromcontrol by protease factor I and its cofactors. Associationof C5(0¢, 115 kd;/3 75 kd) with surface boundC3ballows the splitting of C5by the C3/C5convertases to yield the anaphylatoxinand chemotacticfactor C5a(9 kd) and large fragment C5bwhich, withoutfurther proteolysis, initiates the self-assemblyof the C(5b-8)C9n(n = 1-18) complexinvolved in membranelysis. Control the activated complementfragments and complexproteases is mediated partly by the intrinsic decayof the C3/C5convertases,partly by interaction with a variety of control proteins, and partly by specific degradationby proteases(Table1).

Annu. Rev. Immunol. 1988.6:161-195. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Annual Reviews

164 CAMPBELL,LAW, REID & SIM

Annu. Rev. Immunol. 1988.6:161-195. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Annual Reviews COMPLEMENT SYSTEM GENES

165

Annual Reviews

166

CAMPBELL, LAW, REID & SIM

Annu. Rev. Immunol. 1988.6:161-195. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

THE C1 COMPLEXAND CI-INHIBITOR The Clq moleculeis composedof six globular "heads," each joined by a collagen-likeconnectingstrand to a fibril-like central portion. Eachof the three types of chain (6A, 61~, and 6C) found in the moleculecontain terminal collagen-like sequences, and these combineto form the triple helixes whichmakeup the connectingstrands andfibril-like central portion (2). Each of the "heads" is composedof the C-terminal regions of one A-, one B-, and one C-chain. The Clr2Cls2 Ca2÷-dependentcomplexis consideredto interact with the collagen-like connectingstrands of Clq in such a mannerthat the catalytic domainsof Clr and Cls are closely associatedwithin the "cage"of collagen-like strands (2). Cl-Inhinteracts reversibly with the native unactivatedC 1 complexpreventingspontaneous activation of Clr within the complex.This inhibitory effect is overcome by efficient activators of the classical pathway,such as immune complexes, while activation by nonimmune substances such as DNAis impaired (2). Over-activationof the systemis prevented by the rapid removalof activated Clr and Cls from the C1 complexby Cl-Inh.

Clq A cDNA clone for the B chain of humanClq was isolated from a liver library (4). SubsequentNorthernblot analysis indicated that high levels of Clq B-chain mRNA are found in cultured monocyteswhile relatively low amountsare seen in liver, and no evidencefor significant synthesis couldbe seen in a variety of other tissues. Thefinding of cDNA clones for the A chain of humanClq in a monocytecDNA library (5) along with the demonstration of the synthesis, by cultured monocytesand macrophages,of a C 1 q moleculeapparentlyidentical to serumC1 q (6, 7) together indicate that macrophagesmaybe a major source of serumClq. The Clq B-chaingene is approximately2.6 kb long (4) and has been assigned chromosome 1 p, as has the A-chaingene (5). The B-chaingene contains a single intron of 1.1 kb locatedwithinthe codonfor glycineB-36,precisely the position wherethe triple helical chains of Clq appear to bend when viewedin the electron microscope.Thus,it is of someinterest to see if the A- and C-chaingeneswill also havean intron at the equivalentposition. Twotypes of C1q deficiency havebeendescribed, both of whichlead to the developmentof immune-complex related disease. In one type, a nonfunctional formof Clqwhichhas antigenic activity is found;in the second type no functionalor antigenicactivity is seen. In those with the first type of deficiency, the B chain appears normalas judged by genomicSouthern blotting. The sequenceof the B-chaingeneisolated fromthe library of a patient having the second type of deficiency showedthe presence of a

Annual Reviews COMPLEMENTSYSTEM GENES

167

single base changewhichgenerateda termination codonwithin the globular region of the chain at residue B-150.Thus,in this particular case the deficiency appears to be a consequenceof inability to synthesize the B chain or to secrete a moleculewith a truncated formof the B chain.

Annu. Rev. Immunol. 1988.6:161-195. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Clr and cDNA clones for the Clr and Cls proenzymeshave been isolated from a humanliver cDNAlibrary, and their amino acid sequences show 40% overall homology. In the activated form of both enzymes, an A- or noncatalytic heavychain of approximately55 kd originates fromthe amino terminal end of the proenzyme.It contains an LDLreceptor type B domain and two different pairs of internal repeating sequences,followedby a Cterminal B chain of approximately27 kd whichcontains the active site characteristic of serine proteases (8, 9, 10). Thefirst set of repeating homologyunits (occupying approximately positions 10-80 and 185-255) in the A chains does not appear to have counterparts in other known protein sequencesand thus maybe involved in the activation and control of specificity of Clr and Cls. The LDLreceptor type B (EGF-like) (see Figure 4) domainis located betweenthese first two repeating units. The second set of repeating homologyunits, each of approximately60 amino acids and occupyingthe C-terminal portion of the A-chain, is homologous to the 60-aminoacid repeat (SCR:short consensusrepeat), whichis widely found in the C3b/C4bbinding proteins such as C4bpand factor H (see later section concernedwith the occurrence of homologousunits within the aminoacid sequencesof complement proteins). TheC1r 2C1 s2 complex therefore contains 8 of the 60-aminoacid repeat units, probablylocated near the outsideof the "cage"of the collagen-likeconnectingstrands of the Clq molecule.It is possible they could interact with C4. Boththe Clr and Cls genes lie in the region p13 on humanchromosome 12. Studies using pulsed-field electrophoresis techniqueshaveindicated that the genes are tightly linked; both are containedwithin a stretch of DNA of about 50 kb (9).

C l-Inh Description of the cloned DNAfor Cl-Inh and its use in the study of patients with hereditary angioneuroticedemais given by Dr. A. E. Davies in another chapter of this volume.

THE HLA CLASS III

COMPLEMENT GENES

Phenotypicgenetics haveestablished that C2, factor B, and C4are polymorphic (11). Encodedby two closely linked loci (C4Aand C4B)C4is

Annual Reviews

Annu. Rev. Immunol. 1988.6:161-195. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

168

CAMPBELL, LAW, REID & SIM

exceptionally variable, with morethan 35 alleles described for the two isotypes (12). Thegenes encodingthese proteins mapto the HLA class III region on the short arm of humanchromosome6 and lie between the HLA-Dand HLA-Bloci (13). In the mousethese genes have been mapped to the S region of the H-2 complexon chromosome 17 (14) and also the MHC of a numberof other species (11). The isolation of cDNA clones for C2, factor B, and C4 from liver libraries (15) has allowedthe isolation and characterization of cosmids containing the correspondinggenes (16). The C2and factor B genes are 421 bp apart (17) and lie about 30 kb from the C4Alocus, which is separated from the C4Blocus by ~ 10 kb. Further mappingof the cosmid clones identified a gene encoding the cytochromeP-450 21-hydroxylase (21-OHase),-~ 3 kb from the 3’ end of each C4gene (18, 19). Southern. blot analysis of individuals with 21-OHasedeficiency and DNA sequencing of both genes have shownthat the 21-OHaseB gene is the active gene, while the 21-OHaseA gene is a highly homologouspseudogene(20, 21, 22). Asimilar organizationhas also beenestablishedfor the complement and 21-OHasegenes in the S region of the mouseH-2 complex(14). The single C2 and factor B genes were found to be closely linked and to lie ~ 50 kb from two C4-1ike genes separated by ~ 80 kb. It was later established that the gene closest to factor B encoded Sip. Twogenes encoding 21-OHasewere also placed immediately 3’ of the Sip and C4 genes, respectively; in contrast to the situation in humans,however,it is the 21-OHaseA gene that is important in steroid biogenesis (23). The powerfultechnique of pulsed field gel electrophoresis, whichcan separate large DNA fragmentsup to 2000kb (24), has been used recently to determinethe orientation and position of the complement genes in the HLA class tli region (Figure 2). Linkageof the DR-r, DR-~,and C4genes wasdefined in a large Not I fragment~ 920-980kb in size (25, 26, 27). Further studies were done using a newsingle-copy hybridization probe lying ~ 50 kb beyond the 5’ end of the C2 gene and probes for the knownMHC gene loci in Southern blot analysis of genomicDNAfrom a lymphoblastoidcell line (HLA-A2,BT, Cw7,DR2,C2C, Bf S, C4A3, C4B QO)whichhad beendigested with infrequently cutting restriction enzymes and separated on pulsedfield gels. Togetherthese permittedconstruction of a long-range mapof the MHC (27) (Figure 2). This indicated that MHC could span up to 3800kb artd that the class III region spans ~ 1100 kb. The C2gene was shownto lie telomeric to the C4 gene. The DR~and C4geneswerefound to be separated by at least 300 kb. This analysis also firmly established that the genes for tumor necrosis factors ~ and r, previously mappedto the MHC (28), lie in the class III region betweenC2

Annual Reviews COMPLEMENTSYSTEM GENES HOUSE (Ba~W¢I CIossl Class II K

] 69 lOOkb

Closs I

CLass D

I

Oa

TLo

TNF

Annu. Rev. Immunol. 1988.6:161-195. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

HUHAN HLA DPDZ

ClassII DO OX~O

Clms I

Class

I~A-B HtA-C

DR

HI~A

THF

’

sbo

~

~oo’

z~

z~

Figure 2 Molecular map of the HLA-region on human chromosome6 and the H-2 complex on mousechromosome 17. The centromere is to the left and the telomere is to the right. The orientation and molecular mapposition of the complement genes has been determined for a haplotype with single C4Aand 21-OHase B genes. The position of the 21-OHase A and C4Bgenes on normal haplotypes is shownby the in~et. The HLA-Aloci at the right of the figure is shortened for convenience but could span up to ~ 1000 kb. The information for the mapsis taken from (27), (28), and (161).

and HLA-B.The TNFagene lies 390 kb telomeric of the C2 gene and about 250 kb centromeric of the HLA-Blocus. A similar analysis of the H-2complexhas revealed that the orientation of the complement genesis the sameas that in humans(29) (Figure 2). addition the distances betweenthe C4and Eg genes and betweenthe C2 and TNFggeneswere comparable,430 kb and at least 420 kb, respectivelythough the TNFgeneswere only 70 kb from the H-2Dlocus. The physical distances betweenthe complement genesand the flanking class I and class Hloci in both humanand mouseare sufficiently large to accommodate a numberof as yet unidentified genes.

C2 TheC2mRNA of 2.9 kb directs the synthesis of a 754 aminoacid primary translation product whichyields a plasma form of C2composedof 734 aminoacids (30). Astriking feature whichC2shares with certain other complementand noncomplement proteins is the presence of three contiguous internal repeats, each of about 60 aminoacids, termed SCRs, whichare found at the N-terminusof the molecule(see Figure 4). Characterizationof the geneencodingC2has shownthat it spans 18 kb

Annual Reviews

Annu. Rev. Immunol. 1988.6:161-195. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

170

CAMPBELL, LAW, REID & SIM

of DNA (31), though as yet the exon-intron organization has not been reported. In individuals with C2 deficiency, whichis one of the most commoncomplementdeficiencies found in humans(32), Southern blot analysis has suggestedthat the deficiency is not due to genedeletion or rearrangement(33). Northernblot analysis of peripheral blood monocyte RNApreparations from C2-deficient individuals revealed no detectable C2 mRNA. This suggests that C2 deficiency mayresult from a defect in transcription of the gene or from posttranscriptional processing of C2 mRNA. Althoughpolymorphism in C2 has been detected by isoelectric focussing, the techniquehas beenrelatively uninformative,as mostindividuals are homozygous for the common variant C2C(gene frequency 0.97) (11). However,the use of specific cDNA and genomicprobes in Southernblot analysis of genomicDNA revealed a numberof RFLPsthat subdivide the C2Callele (31). Whenthe enzymeSst I and a 300 bp genomicprobe were used, an interesting polymorphism, whichmapsto a region near the 5’ end of the C2gene, wasdefined. It is multiallelic with the probehybridizing to Sst I restriction fragmentsof 2.7, 2.65, 2.6, and2.4 kb. Lengthvariation of other restriction fragments(BamH I, HindIII) has been observedusing this probe, indicating that the Sst I RFLPis due to the insertion or deletion of short sequences. This polymorphismsubdivides previously indistinguishable haplotypescarrying the C2Cand factor B F alleles (31) and also appears to subdividesomehaplotypescarrying the C2Cand factor B S allele (S. J. Cross, R. D. Campbell,unpublished). The cloning of mouseC2 cDNA has recently been reported, and this has been used in Southern blot analysis to define polymorphismin the murineC2 gene (34). Factor B Humanand mouse factor B cDNAhave been cloned (15). The 2.6 humanfactor B mRNA directs the synthesis of a 764 aminoacid primary translation product whichyields a plasmaform of factor B composedof 739 aminoacids (30). The factor B geneis 6 kb long andis split into 18 exons(35). Three these exonsat the 5’ end of the geneexactly encodethe three SCRswithin the Bafragmentthat showsignificant homology to similar repeating units in a numberof other proteins (see later section concernedwith the occurrence of homologous units within the aminoacid sequencesof complement proteins). Theeight exonsin the 3’ one third of the geneencodethe serine protease domain.Thus, factor B and also C2 are membersof two distinct families of proteins (see later sections), and probablyarose by the fusion of at least two gene segmentsof distinct evolutionary origin (30). The

Annual Reviews

Annu. Rev. Immunol. 1988.6:161-195. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

COMPLEMENTSYSTEM GENES

171

homologyin structure and function between C2 and factor B proteins and the close linkage of the genes suggests that they arose from an ancestral gene by duplication. However, although the mRNA molecules encoding C2 and factor B are similar in size (2.9 kb versus 2.6 kb), the C2 gene approximately 3 times longer than the factor B gene. Factor B, like C2 and C4, is polymorphic, and variants have been defined by differences in charge. There are two commonalleles F and S (gene frequency 0.27 and 0.71), two less commonalleles Ft and St with gene frequencies of about 0.01 each, and up to 14 very rare alleles (11). The two commonalleles F and S have been sequenced (35). Comparison over ~ 7 kb, including the 5’ and 3’ flanking regions of the gene, revealed only six differences of which two lie in exons. One is a silent mutation in the third position of the codon for Tyr-199 and results from a C -~ T transition. The second changeis a G --, A transition in the secondposition of the codon for amino acid 7 of the mature polypeptide. This results in an aminoacid change from Arg in the S allele to Glu in the F allele. This wouldfit with the difference in electrophoretic mobility of the two variants, with the Fallele carrying less positive charge and thus movingmore toward the anode. RFLPshave also been defined using the factor B cDNAprobes (31). An MspI polymorphism due to the nucleotide difference in codon 7 is informative for the F and S alleles, while two others detected using Rsa I and Taq I are found associated with only someof the F alleles (31). The major site of synthesis of factor B and C2 is the liver, though cells of the monocyte/macrophageseries synthesize them at extrahepatic sites (36). DNAsequencing, S1 mapping, and primer extension experiments have established that the transcription initiation site of the factor B gene lies only 421 bp from the poly (A) site of the C2 gene (Figure 3) (17). Despite this close linkage, the genes for factor B and C2 appear to be regulated independently in different cell types. Interleukin-1 will increase

-1000 -800

~

’

-600 -~0

’

c~

-200

1 (bp)

’

FMo~B

pol¥~ sire

m RN~

AATA~ nhoncer = 0 TATABox | Enhoncer core

TATA -’=~---I}-

Promoter Interferon concensus Enhnncer mofifs

Figure 3 Close linkage of the C2 and factor B genes. The polyadenylation site of the C2 gene lies 421 bp from the transcription start site of the factor B gene. Also shown are the control elements necessary for the constitutive and inducible expression of the factor B gene. The cell-specific expression of the factor B gene is due to the combinatorial effect of the promoter and the enhancer elements (17).

Annual Reviews

Annu. Rev. Immunol. 1988.6:161-195. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

172

CAMPBELL, LAW, REID & SIM

expressionof factor B in hepatocytesbut has no effect on the expression of C2 (37). Althoughinterferon-7 can induce both factor B and synthesis, the kinetics of C2 induction in primarycultures of human monocytes are different fromthose of factor B (38). Inspectionof the Y-flankingregionof the factor B gene has revealed the presenceof several sequencesshowingstriking homology to sequences thoughtto play a role in the regulationofgeneexpression.Thesesequences are shownin Table2 andhighlightedin Figure3. At nucleotides -28 to -25 is a TATA box. At nucleotides -234 to -227 is the sequence GTGGTTTG, which matches the consensus core motif of an enhancer elementof the SV40type. Nucleotides -154 to -127 contain a 28 bp sequenceclosely homologous to a commonsequence (interferon response sequence--IRS) foundin the promoter region of several genes responsiveto IFN-a(39). Table 2 Homologies found in the factor B Y-flanking region with knownregulatory sequenc~ (from ~ 17) Sequence TATABox consensus

G-

GTATATAA~

Factor B

TTGTATAAAAGGCTG --31

-G-

-G

o o o o o o o o o

TTC

Factor B

GGTGGGACTTCTGCAGTTTCTGTTTCC" -154

Factor B

MCMVenhancer18bp consensus

~ -

--17

a-Interferon consensus

Enhancercore consensus

-

o

o o

ACCTC-

o o

0 o o

GCAGTTTCTCv

o o

o 0 o

c o o

TCTC"

o

-1~

o o o o o o o o

GTGGTTTG --234

--227

A TCAATAGGGACTTTCCAx o

o o o o o

o o o o o o

TTCACATGGAATTTCCCA --472 HCMVenhancer19bp consensus

o o

--455

CACCATTGACGTCAATGGG o o o

o o o o

o o o o

GACCTTTGGCAGCAAAGGG -- 171

o o

-- 153

a Pointsindicatenucleotideshomologous with the concensus sequence.Coordinates for the FactorB sequencesa: givenrelativeto the CAP site. Adashindicatesanynucleotide.

Annual Reviews

Annu. Rev. Immunol. 1988.6:161-195. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

COMPLEMENT SYSTEMGENES

173

In a transient assaysystemthe reporter genechloramphenicol acetyl transferase (CAT) under control of the factor B 5’ flanking region could be induced1.g-fold by IFN-~(17), and IRSwasnecessary for this induction (40). Deletion analysis of the factor B Y-flankingregion has suggestedthe presenceof cis-acting DNA elementsthat are essential for the cell-specific expressionof the factor B gene(17). Atranscriptional enhancer,whichacts in a position- and orientation-independent manner,was defined between nucleotides - 496 to - 201. In addition, the promoterwasfound to extend up to 260 bp fromthe factor B CAPsite. The cell-specific expression of the factor B gene maybe dependentuponthe combinatorialeffect of the promoterand the enhancer. Thefactor B enhancer,althoughat the 5’ end of the gene, is also at the 3’ end of the C2gene. Thedefinition of an enhancerin the 3’ region of a geneis not unique(41). However,as the C2promoterlies 18 kb away, remains to be established whether the factor B enhancer is capable of activating transcription over sucha large distance. This maynot be necessary as a transcriptional enhancerhas also beendefinedin the 5’ flanking region of the C2gene (L. C. Wu,R. D. Campbell,unpublished).

C4 In humansthere are two C4 genes, C4Aand C4B, which lie about 10 kb apart and which are transcribed into mRNA molecules of 5.5 kb. Comparison of the C4Aand C4BcDNAsequences has revealed < 1% nucleotidevariation. Of the 14 nucleotidedifferences, 12 are clustered in the C4dregion and cause 9 aminoacid substitutions (42). The sequences of 7 further C4Aand C4BcDNAand genomic clones (43, 44) have established the pattern of polymorphismin the C4d fragment (44) and provideda structural basis for the observedelectrophoretic, serological, and functional differences betweenthe isotypes. Of importancein this respect wasthe cloning and sequenceanalysis of two unusual C4alleles, C4A1 and C4B5 (44), whichpossesstheir ownclass specific properties (45) but have essentially the reversed Rodgers (Rg) and Chido (Ch) antigenicities (46). Comparison of the available sequencesallowedthe location of the two Rgand six Chdeterminantsto be deduced(44). In addition the sequencecomparisonhas helped to define the four isotypic aminoacid differences betweenC4Aand C4Bthat are responsible for their different chemicalreactivities (Table4). C4genescan differ in size (44, 47, 48), Thus,C4Bgenes can be either 22 kb or 16 kb, due to the presence or absenceof a 6-7 kb intron about 2.5 kb from the 5’ end of the gene (44). In a population study long C4 genes included all C4Agenes studied and also someC4Bgenes, e.g. C4B1

Annual Reviews

Annu. Rev. Immunol. 1988.6:161-195. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

174

CAMPBELL, LAW, REID & SIM

on most A3B1haplotypes. Similarly, C4Bnull alleles (C4BQO) were found to be 22 kb in size. Of the remainingC4Balleles, approximatelyhalf were of the short form(47, 48). Differenceshave also beenobservedin the numberof C4genes expressed (49). Duplication of C4Bas C4B1,C4B2on the extended haplotype B14 C2CBf S C4A2,C4B1,C4B2,DR2has been demonstrated at the protein level (50) and has also been observedfrom cosmidcloning wherethree C4 genes, one C4Aand two C4B, were found on one haplotype (51). unusual phenomenon in the genetics of C4is the high frequencyof null alleles at either locus (C4AQO, 5-15%;C4BQO, 10-20%)(52, 53) defined by the absenceof C4Aor C4Bin plasma. Theyare of particular interest as they occur at a greater frequencyin individuals with someHLA-related diseases such as SLE(54). In a recent study it was found that ~ 60% the C4haplotypescarrying a null allele weredue to deletion of the gene usually together with the flanking 21-OHase gene(47, 55). Unequalcrossover during meiosis would explain duplication on somehaplotypes and deletion on others (56). However,in other situations wherethe gene present but not expressed, the molecularbasis of the QOallele has not beendefined. In somecases this maybe due to defects in transcription or translation. However,by the use of RFLPsdefined by N1a IV and Eco0109 whichrepresent the exact locations responsible for isotypicity between C4Aand C4B,and their generally associated major Rodgers (Rgl) and Chido(Chl) antigenic determinants,respectively, it wassuggestedthat the null allele on the HLAhaplotype B44 C2CBfF C4A3, C4BQO DR6may not be a C4Ballele but probably encodes another C4A3allotype at the secondC4locus (57). A similar situation also is apparentwith respect some of the homoduplicated C4 genes, e.g. C4A3C4A2C4BQO or C4A5 C4A2C4BQO, whereit has been suggested that both loci are present as normal, but that the second(or C4B)locus encodesa C4Aprotein (58). A numberof RFLPshave been defined at the C4loci (see 15, 56, 57). Of particular use are the TaqI polymorphicpatterns specific for the 5’ end of the C4loci and for the neighboring21-OHase loci, and the Nla IV and Eco0109polymorphismsdiscussed above. In a combined genomic Southernblot analysis the RFLPscan define the nature and characteristics of the C4loci (57). In mousethe two C4-1ikegenes encodingC4and Sip are both 16 kb in size (14). However,the two isotypes are not as conservedas the two human isotypes and share only 94%homology (59, 60). AlthoughC4and Sip share a numberof structural and biochemicalfeatures, including immunological cross-reactivity, a heterotrimeric structure, biosynthetic pathways,and an internal thioester bond,still Sip is not activated by Cis, dueto a cluster of differences aroundthe Cis cleavagesite (59, 60). Sequencecomparison

Annual Reviews

Annu. Rev. Immunol. 1988.6:161-195. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

COMPLEMENTSYSTEM GENES

175

of Slp froma numberof mousestrains reveals that nucleotidechangesthat alter aminoacid sequence(replacementsubstitutions) are accumulating the samerelative rate as silent substitutions, andthis has beentaken to suggestthat SIp has no functional role (61). In most mousestrains C4is constitutively expressedby the liver while the Slp geneis inducedby testosterone or not expressedat all. Nonakaet al (62) havesuggestedthat the difference in expressionof C4and Sip may be due to a positive regulatory domainlocated betweennucleotides - 1700 to -400 from the transcription initiation site. However,in somewild derivedstrains Sip is constitutively expressedin the samewayas C4. In a strain expressingSip constitutively, it wasfoundthat the geneis a C4-SIp recombinantin that it comprisesa 5’ region derived from a C4gene and a 3’ region derived from an Sip gene (63). A biological advantage for the constitutive expressionof Sip is not clear as Sip has no knownfunction. Similar to mouse,humanliver is the major site of synthesis of plasma C4, and the expression is constitutive. Wuet al (64) have defined the presence of several cis-acting DNA elements within 1 kb of the transcriptional start point. TheC4genecontainsa relatively strong andcompact non-TATA box promoter within nucleotides -147 to + 13, and also a transcriptional enhancer. However,the regulatory elements, in the 5’ flanking DNAseem to be rather complex, with a relatively compact positive regulator within < 200 bp which includes also the promoter and the CAPsite, and a distal positive regulator separated by a negative regulator. C3, C4, and C5 Complete eDNAand derived amino acid sequences are available for humanC3(65), C4(42, 43), and mouseC3 (66, 67), C4(68, 69), (70). Partial eDNAclones for humanC5 (71) and rabbit C3 (72) also been reported. Significant homologyhas been found betweenthese proteins. C3, C4, and C5 within each species showpairwise identity of about 25 %after alignment.Thesameprotein fromdifferent species shows 75-80%identity. Thegene structure and organization for C4have been covered in the previous section. The genes for humanC3and those for mouseC3 and C5 do not lie within their respective MHC complex. The humanC3 gene was mappedto chromosome19 (73), the mouseC3 gene to chromosome 17 but about 12 cMfrom the H-2region (74, 75). Byisolating overlapping genomieclones and hybridizing with different eDNA fragments, the size of the mouseC3gene was estimated to be 24 kb (76). The mouseC5 gene was located on chromosome 2 (77).

Annual Reviews 176

C~,M~’BEI~L, LAW,REID & SIM

Annu. Rev. Immunol. 1988.6:161-195. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Biosynthesis

and Processin#

c3, C4, and C5are synthesized as single polypeptides before they are secreted/processed;this includes the removalof the signal peptide, glycosylation,sulphation,synthesisof the internal thioester, andthe tailoring of the moleculesby proteolysis of the single-chain promoleculesto their final multichainstructures. Acomparisonof the deducedprimarystructure with aminoacid sequencesobtained by peptide sequencing,especially the N-terminalsequencesof the native 0~,/~ (and ~) chains, showedthat the chains are aligned in the order of/~-e in C3 and C5 and /~-e-~ in C4 promolecules. Furthermore, the N-terminal sequences of the e (and 7) chains in each case are precededby four residues, at least three of which are basic (42, 59, 60, 65-71, 78-82). This type of processingis also found in factor I (83, 84) (see Table The Internal Thioester and Covalent Bindin# Reaction C3 and C4 each have an internal thioester that enables them to bind covalently to cell surfaces and immuneaggregates upon activation. The thioester is locatedmidway in the respective promolecules,andit is between the cysteine and glutamineresidues in the sequence-Cys-Gly-Glu-Gln(see 85). This thioester is also foundin ~2-macroglobulin and related proteins (86). Howthe thioester is synthesizedis not known.In a cellfree system, Iijima et al (87) demonstratedthe biosynthesis of inactive C3fromrabbit liver mRNA and rabbit reticulocyte lysate. Asdetected by its ability to incorporate methylamineto its thioester, active C3could be generated fromthe inactive product by the addition of liver homogenate. It appears that a factor, possibly an enzyme,is required for the formation of the thioester. C5does not havean internal thioester and does not bind covalently to cell surfaces. Alignmentof the mousepro-C3and pro-C5shows that the -Cys-Gly~Glu-Glnstretch in C3is replaced by -Ser-Ata-Glu-Alain C5. The Ser -~ Cys and Ala ~ Gin substitution clearly could account for the absenceof the thioester in C5(70). Theprimarystructure of the thioester andthe adjacent residues of C3, C4, and C5, as well as of related proteins, are shownTable4 (42, 59, 60, 65, 67-70, 72, 88-90). Althoughthe covalent binding reaction wasfirst describedfor C3(91), muchof the subsequentworkto characterize the chemicalspecificity was done on C4. This is becausethe two isotypes of humanC4, whichdiffer by four aminoacid residues, have markeddifferences in their reactivity with aminoand hydroxyl groups. At neutral pH, C4Areacts exclusively with amino groups, whereas C4Breacts comparably with amino and hydroxylgroups,(92, 93). It is possibleto correlate the bindingspecificity to the four aminoacids that.are characteristic of the isotypes (44, 45). The

Annu. Rev. Immunol. 1988.6:161-195. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Annual Reviews COMPLEMENT SYSTEM GENES

177

Annu. Rev. Immunol. 1988.6:161-195. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Annual Reviews 178 CAMPBELL, LAW, REID & SIM

Annual Reviews

Annu. Rev. Immunol. 1988.6:161-195. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

COMPLEMENTSYSTEM GENES

179

four residuesare about100 aminoacid residues C-terminalto the thioester; they are -Pro-Cys-Pro-Val-Leu-Asp-for C4Aand -Leu-Ser-Pro-Val-IleHis- for C4B.Sequencesinclusive of these putative catalytic residues of the humanC4isotypes, as well as the correspondingsequencesin related molecules,are also shownin Table 4. It is interesting to note that the correspondingsequencein mouseC4 (and Sip) is a hybrid betweenhuman C4Aand C4B,the residues being -Pro-Cys-Pro-Val-Ile-His-.Preliminary data indicate that its bindingspecificity is similar to that of C4B(94). This mayput a further limitation on the relevant residues to the Leu-Asp/IleHis exchange.It is not clear howthese residues confer specificity to the binding reaction though one can speculate that the Aspresidue in C4A deprotonizes aminogroups to enhancetheir nucleophilicity and the His residue in C4Bhas a similar effect on hydroxylgroups. However, this does not explain whyC3 with a corresponding sequence of-Asp-Gly-Pro-ValIle-His- has low reactivity to aminogroups.Clarification will require more worksuch as the site-specific modificationof the protein and analysis of the three-dimensional structure of the thioester sites. FACTOR I, ITS COFACTORS AND RELATED PROTEINS Breakdown of the complementactivation fragments C3band C4bis regulated by a numberof control "cofactor" proteins, whichare thought to form a noncovalent complexwith C3b or C4b. C3bor C4bwithin such a complexis then cleavedby the control protease factor I. Factor I The circulating form of factor I is a serine protease consisting of two disulphide-linkedpolypeptidechains (46 kd and 39 kd). Theenzymicactive site is containedin the light chain. Factor I is synthesized in liver and by monocytes(95). ThemRNA size in liver is 2.4 kb (83) andsequencingof factor I cDNA clones (83, 96) shownthat the protein is encodedas a single polypeptide chain of 565 aminoacids, with an additional 18-residue leader sequence. Thesingle chainformis processedmainlyintracellularly (96), but a processingdefect has beenobservedin HepG2cells, resulting in secretionof the single chain form(96). Thesequenceof the light chain is typical for a serine proteinase of arginyl specificity. It has an extra disulphidebridge, absentin mostserine proteases but present in tissue plasminogen activator (tPA) andurokinase. The heavy chain sequencecontains several recognizable homologyunits

Annual Reviews 180

CAMPBELL, LAW, REID & SIM

Annu. Rev. Immunol. 1988.6:161-195. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

(Figure4), includingthe cysteine-richunits known as LDL receptorrepeats class A andB (97). Oneclass B repeatis foundat position 12-59(Figure 4)7 andtwo class Arepeats occur at positions 204-238and239-275. It has beensuggested(96) that factor I containsa factor H-like SCR,but agreementwiththe consensussequence(Figure4) of SCRsis poor. Factor I is a goodexample of a "mosaic" protein, discussedby Doolittle (98). FactorI has twomainpolymorphic variants in the Japanesepopulation (99), but polymorphism has not beendetectedin otherracial groups.A few Ancesfrc~ 60omino ~cidsfructure Serine proteasedomain

C~bi~ingp~tein

~

I

{ A,B )

I

Facfor ~

CRI

p~otdns. The 60 aminoacid~epeati~l ~t~uctu~e (SCR:~), typical o~ C4bpand£aclo~~, o~u~in DAP, CRI,CR2 (nots~o~n),£acto~B, C2, CI~,~nd Cls, as w~llas in noncomplemeni p~otelns IL-7~e~ptoe, ~I,~aclo~XIIIJ s~bunit, andhapto£lobln 2 (I02,I03, 123).The co~e~s~ s~q~n~o~ theseunitsis:

Sefine proi~as~ units (~ioa] o£~.~, t~ypsin o~u~in C2 andB, C IL Cls,~acto~ I, a~d~ac~o~ D. LDLr~o~pto~ ~epeat ~t~uctu~es typ~A (97)occ~in Faclo~ I (83,96)andp~ffo~n, C7, and9; as wallas in LDLle~pto~. Thewidespread DDL~ecep~or classB ~epeat(somefim~ call~dan ~O~domain) is i~ ~acto~I; C7,8, and9; CI~ andCls;and alsoin coalulation p~o(eins IX, X, a~d C, tPJand u~okina~e, and~O~,TOF and~O~ (83,97,98).DiaTonaHy shaded sections ~p~esent se~uence~ un~dat~d to thedomains discussed above.

Annual Reviews COMPLEMENTSYSTEM GENES

181

cases of factor I deficiencyhavebeendescribed,and inheritanceappearsto be autosomal codominant(100). The gene for factor I has been mapped to humanchromosome 4 by the somatic cell hybrid method(96).

Annu. Rev. Immunol. 1988.6:161-195. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Factor I Cofactor Proteins and Their Homologues Factor H(H), C4bp,CR1,and MCP(formerly called gp45-70), all act cofactors for the factor I-mediated breakdownof C3bor C4b(for review see 101-103).Theseproteins differ in size (Table1), but primarystructure studies have shownthat H, CR1,and C4bpare highly homologous,each having a repetitive aminoacid sequenceconsisting of contiguousunits, each about 60 aminoacids long, with a frameworkof 4 conservedcysteine residues, and conservedPro, Trp, Gly, and other residues (Figure 4). The homologous 60-aminoacid long units have beenreferred to as SCRs(104). Twoother C3b/C4bbinding proteins, DAFand CR2,have this type of structure, although they lack cofactor activity. Analysisof polymorphic variants indicates that the structural genes for H, CR1,DAF,and C4bp are closely linked (101-103, 105-107). Studies using cDNA probes have shownthat CR1,C4bp, DAF,CR2,and H structural genes are on human chromosome 1 (102, 103, 105). CR1and CR2have been localized to lq32. H and C4bpgenes have also been mappedto mousechromosome 1 (103, 108). Physicalmappingstudies using pulsedfield gel electrophoresis(109) have identified a single 950 kb humangenomicDNA fragmenthybridizing to CR1, CR2, C4bp, and DAFcDNA probes, but not to H probes. This fragmentcan be separated into two pieces, one of 500 kb, containing DAF and C4bp genes, and one of 450 kb, containing CR1and CR2. These studies have identified a major gene cluster, nowknownas the RCA (regulation of complement activition) cluster (107). Factor H H is the most abundant cofactor protein. It is a single chain plasma glycoprotein (155 kd). Humanand mouse H share 61%amino acid sequenceidentity and are madeup entirely of SCRs,of whichthere are 20 in each protein (110, 111). Individual SCRsshow25-45%sequenceidentity with other SCRswithin H. Theconsensussequenceof such units is shownin Figure 4. The structure/function studies of Alsenzet al (112) indicate that the C3b-binding site is likely to be within SCR4 and/or 5. Northern blotting of liver poly A÷ mRNA shows three H-related mRNA transcripts (110, 113). The mRNA corresponding to the main 155 kd plasma protein is 4.3 kb (containing 3.9 kb coding sequence). mRNA species of 5.3 kb has also beenobserved(110), but it is likely be an artefact related to the 2° structure in the 4.3 kb species. A1.8 kb mRNA, which is recognized only by probes from the 5’ end of H cDNA,

Annual Reviews

Annu. Rev. Immunol. 1988.6:161-195. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

182

CAMPBELL, LAW, REID & SIM

also occurs. It is equal in abundanceto the 4.3 kb transcript (110). eDNA clones corresponding to the 1.8 kb mRNA have been shown to encode the leader sequence and the first 427 aminoacids (7 SCRs)of the "full-length" H sequence, followed by: (i) a coding sequence for four amino acids not present in the full length sequence; (ii) a TGAstop codon; and (iii) untranslated region unrelated to the 4.3 kb mRNA (103; 110, 113). The 1.8 kb mRNA is likely to arise from alternative splicing and appears to be translated. Twolow-abundance protein species of 45-50 kd are detected by SDS-PAGE and Western blotting of nonreduced plasma samples, using monoclonalantibodies specific for epitopes with the first six SCRsof H. It is likely that the larger of these two protein species is a translation product of the 1.8 kb mRNA,which has coding sequence for a 49 kd polypeptide (110). The smaller product may be a cleaved form of the larger one. Both proteins are distinct in size from the common proteolytic products of H. The function and activity of the truncated forms of H are unknown, although they contain a sequence corresponding to the C3bbinding site and thus maypossess cofactor activity. A further minor mRNA species of 1.2-1.5 kb is detectable in liver (110). It is recognized only by probes derived from the 3’ end of the H cDNA, and its origin is unknown. Northern blotting of mouse liver mRNA with mouseH probes also reveals 3-4 transcripts (114). Preliminary analysis of the humangene (115) suggests it is large (> kb) and that a further related gene or pseudogenemay be present. In the mouse, there appear to be an H gene of about 90 kb and at least two Hrelated genes or pseudogenesof 60 and 120 kb (114). Humanfactor H has five allelic charge variants (106). A Tyr-~ His exchange in SCR7 of H occurs at the DNAand protein level and probably represents a difference between the two most commonalleles (110). A Bgl II RFLPhas been observed (115). Two cases of complete H deficiency in humans have been described (116, 117), but the defect at the DNAlevel has not been identified. In one case a truncated H protein maystill be expressed (116). C4bp C4bp, like H, is an abundant plasma glycoprotein. It consists of a disulphide-linked assembly of seven identical polypeptide chains, each about 70 kd (101-103). The structure has been examinedby low angle scattering and electron microscopyand is thought to consist of seven relatively rigid rods connected to a base domain(118). It is synthesized in liver. The chains of human C4bp are composed of 549 amino acids, with an additional leader sequence of 13 to 40 residues (119, 120). The N-terminal 491 residues

Annual Reviews

Annu. Rev. Immunol. 1988.6:161-195. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

COMPLEMENTSYSTEM GENES

183

are in the form of eight SCRs,while the C terminal 57 residues do not fit this pattern. Structure/function studies suggest that the C4b-bindingsite is likely to be in the fourth SCRand that interchain disulphide bridging occurs through the C-terminal nonhomologousportion of the polypeptide (103, 121). The mRNA size in liver is 2.5 kb, and only one transcript is detectable (l 19). MouseC4bpis different from the humanprotein, because it consists of a noncovalent assembly of identical polypeptides which are only 413 residues long (121). Again, the length of the signal sequence is ambiguous13 or 56 residues. The first 358 residues are arranged in six SCRs,followed by a 55-residue nonhomologoussequence. This C-terminal stretch lacks the Cys residues thought to be involved in interchain linkage in the human protein. Alignmentstudies suggest that SCRs5 and 6 of the humanprotein are absent in mouse (121). The human and mouse sequences show 52% identity when appropriate regions are compared. The mRNA size in mouse liver is 1.8 kb (121). The human C4bp gene is about 30 kb long, and the mouse gene 20-25 kb (108, 122). There is no indication of additional C4bp-related genes pseudogenes. HumanC4bp is polymorphic, showing three charge variants 006). Althoughfour possible polymorphicsites were identified in sequencing of C4bp(119), their relationship to knownvariants is not established. A Bgl II RFLPhas been described (122). CR1 and

CR2

CR1is a large single polypeptide chain, membrane-associatedglycoprotein present on most leukocytes, erythrocytes, and kidney podocytes. CRI is unusual in that it is polymorphicin size. Sizes are given in Table 1 but these are derived from SDS-PAGE and are underestimates (for summary, see 101-104, 123). Peptide and eDNAsequencing of CR1, using material from tonsilar cells, has provided over 80%of the sequence of the most commonof the four allotypes (CR1-A)(104). A sequence of 5.5 kb of eDNA,containing an open reading frame of 4.7 kb, has been reported (104). Sequencing cDNAfrom an alternatively spliced transcript completes this sequence (124). CR1-Acontains at least 33 SCRs, followed by a 25-residue hydrophobic (transmembrane) segment and a nonhomologous C-terminal region (the cytoplasmic domain) (104). The sequence of CR1 is much repetitive than that of H or C4bp. Most of the SCRswithin CR1can be arranged into four groups, each of seven contiguous SCRs. These segments, each of 450 amino acids, are termed long homologous repeats (LHRs), and the degree of homology between LHRsis muchhigher than,

Annual Reviews

Annu. Rev. Immunol. 1988.6:161-195. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

184

CAMPBELL, LAW, REID & SIM

for example, between randomlyselected SCRswithin H or C4bp. Large portions of LHRsshowup to 99%identity (104). This repetitive nature showsclear evidencefor duplicationand conversionevents in the evolution of CR1.Theallelic size variants are likely to differ fromone anotherby loss or gain of the coding sequencefor 1 LHR(1.35 kb, 50 kd of polypeptide). Fromsequence evidence, the true polypeptide Mr of CR1-Ais 245 kd (104). Northern blotting of mRNA from lymphocyteand lymphocyte-derived cell lines with CR1probessuggests that each allele has two transcripts, generally of unequalamounts,possiblydiffering in the length of the 3’ or 5’ untranslated regions. For CR1-A,the major mRNA species is about 8.6 kb; for B, 11.6 kb; for C, 7.3 kb; and for D, 12.8 kb (124). As for a short (alternatively spliced) mRNA encoding the N-terminal 8.5 SCRs of CR1has been detected in HL-60and EBVtransformed cells (125). further 2 kb mRNA hybridizing to 5’ CR1eDNAprobes is observed in tonsil cells (126). CR1is also unusual in that it showsinherited variation in level of expression. The mechanismunderlying this is unknown,but a Hind III RFLPwhichcorrelates with genetically determinedhigh or low expression has beencharacterized (127). CR1 eDNA probes hybridize to CR2eDNA,indicating a dose similarity betweenthe two sequences.CR2is a single polypeptidechain glycoprotein of about 140,000Mr, whichis expressedon B lymphocytes.It serves as a receptor for C3b, iC3b, C3d, and C3dg, and for Epstein-Barr Virus. The CR2mRNA in tonsil B cells is 5 kb in length (128). Peptide and eDNA sequencing studies of CR2eDNA indicate that it is madeup of 15 SCRs followed by a transmembranesegment(24 aminoacids) and a 34-residue cytoplasmic domain(129). CR2is discussed further by Cooperin this volume. HumanCR1eDNAprobes also cross-hybridize to several DNA sequences of mice. As well as candidates for mouseCR1and CR2(130), these include genes (termed X and Y) each of which forms 2 kb mRNA transcripts encoding sequences (SCRs)homologousto CR1(131). These transcripts are found mainlyin nonlymphoid tissue and their function is unknown. Gene X is on mouse chromosome8, gene Y on chromosome one. DAF and MCP DAFis a protein of about 70 kd present on the surface of a wide range of cell types: erythrocytes,all leukocytes,platelets, epithelial cells, and connectivetissue. Thereis a soluble formpresent at low concentrationsin plasma,tears, saliva, and urine (101-103,123). DAFis distinct fromthe other proteins discussedabovein that it is heavily O-glycosylated.Peptide

Annual Reviews

Annu. Rev. Immunol. 1988.6:161-195. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

COMPLEMENTSYSTEM GENES

185

and eDNA sequencing studies have been reported for DAFderived from Hela and HL-60cells (132, 133), and in both cell types several mRNA transcripts are observed.Thesemayarise partially fromdifferent lengths of 3’ untranslatedregion and fromalternative splicing of a 118 bp intron that gives rise to a frameshift near the C-terminus. Theproduct of the longer mRNA (the minortype in Hela cells) is thought to correspond the soluble form of DAF,the sequenceof whichis madeup of 406 amino acids (plus a leader sequence).TheN-terminalregion contains four SCRs, followedby a region rich in serine and threonine, probablyrepresenting the sites of O-glycosylation,and then by a hydrophilicC-terminalregion. The shorter mRNA (lacking the 118 bp intron) is likely to produce 347-residue protein whichdiffers by having a hydrophobicC-terminus, containing a probable transmembranesegment. This form maybe processed further to produce the mature membrane-bound DAFwhich is anchored to the membraneby a glycophospholipid moiety. The single DAFgene is 35 kb long (134). Nosequence data are yet available for MCP,a 58-63 kd glycoprotein with a distributionsimilar to DAF (135). In viewof its functionalsimilarity to the cofactor proteins, it will probablyhavecommon structural features. General Observations on SCR Structure Althoughpresent in manyC3b/C4b-bindingproteins, the SCRstructure is evidentlynot associatedonly with this function since it can be foundin noncomplement proteins (Figure 4). It is likely to represent a structural building block, similar in concept to the immunogloblin fold, onto which sequence variations related to function are superimposed.Fragmentary disulphidebridgingassignmentsare available, and these generallyindicate that each SCRcontains two internal disulphide links, bridging Cys2 to Cys 4, and Cys 1 to Cys 3 (102, 103; A. J. Day, J. Janatova, personal communication). Each SCRis likely to represent a compact domain, probablyconsisting mostly of fl structure. Hydrodynamic data, electron microscopy,and low angle scattering data showthat proteins containing manySCRsare very elongated(H, C4bp,f12I) (136), and it is likely their structures are similar to long strings of beads, eachbeadrepresenting an SCR(dimensions30 x 42.5/~; 118), joined to its neighborby a short linking strand. At the DNA level, fragmentarydata indicate that, in general, each SCR is encodedby a single exon(35, 102, 103), althoughthere are exceptions (SCR2of mouseC4bpand H, and the one in haptoglobin; 108, 114). The evolution and widespreaddistribution of these units is of considerable interest, although the large size of the genes in the RCA cluster is an obstacle to rapid elucidationof the evolutionof the genes.

Annual Reviews 186

CAMPBELL, LAW, REID & SIM

Annu. Rev. Immunol. 1988.6:161-195. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

COMPLEMENTRECEPTOR TYPE 3 (CR3) CR3is distinguished from the other C3 receptors in its divalent-cation dependent affinity for iC3b (for review see 137). It is considered to have lectin-like properties and is found on cells of myeloidlineage as well as granulocytes. Onunstimulated cells, CR3probably plays a supportive role to other receptors, e.g. Fc-receptor, in phagocytic activities. However, the CR3-iC3binteraction is apparently sufficient to trigger phagocytic response whenthe cells are stimulated by phorbol esters (138, 139). CR3 is composedof two noncovalently linked subunits, a and ft. The same flsubunit is also found on at least two other cell surface adhesion antigens, p 150,95 and LFA-1(140). p 150,95 also has a demonstrableaffinity to iC3b (141, 142), but its function maylie in the conjugation between cytotoxic T lymphocytes and target cells (143). LFA-1 mediates various T lymphocyte activities (for review see 144). The fl-subunit of the humanadhesion antigen has been cloned, and the primary structure obtained (145, 146). It has six glycosylation sites, putative transmembrane segment, and a cytoplasmic domain of about 47 residues. The most striking feature is the abundanceof cysteine residues (56 out of 747), 42 of which were found in a span of 256 residues that includes several internal homologyunits. Analysis of the DNAof a limited number of "normal" individuals by Southern blots revealed a polymorphismwith the Bgl II enzyme (147). The gene of the fl-subunit has been mapped to chromosome 21 (148), and it was estimated to have upper size limit of 32 kb (146). Patients deficient in CR3are invariably found to be deficient also in LFA-1and p150,95, leading to the conclusion that it is their fl-subunit that is defective (144). Biosynthetic studies and analysis of their mRNA demonstrate the heterogeneity of their deficiency. At the mRNA level deficiency could be caused by the absence of or a decrease in mRNA, but some of the patients have the normal levels. All mRNA found thus far have the same size, about 3.2 kb. At the biosynthetic level the patients either produce a precursor molecule of aberrant molecular weight or one of normalsize, but in neither case do they matureto a correct final product (149, 150). Twoother molecules, reported to date, have a structure similar to that of the fl-subunit. They are a subunit of integrin from chicken fibroblasts (151) and the subunit IIIa of GPIIb/IIIa from humanepithelial cells and platelets (152), both of which have affinity for fibronectin. The three molecules have a pairwise identity of over 40%, which includes conservation of all cysteine residues. The complete structure of the a-chains of cell adhesion proteins is

Annual Reviews COMPLEMENTSYSTEM GENES

187

not known,but available N-terminal sequences showextensive homology (153). This homology also extendsto the subunitsof the fibronectin binding proteins. Thus, the cell surface adhesion glycoproteins and fibronectin receptors are likely to be membersof the samefamily that have evolved awayfrom each other to mediate more specific functions in different systems(154).

Annu. Rev. Immunol. 1988.6:161-195. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

C5b-9,

PERFORIN, ANDS PROTEIN

It is becomingclear that the componentsof the membrane attack complex (MAC),C6, C7, C8, and C9are structurally, antigenically, and functionally related (3). C6and C7are both single chain glycoproteins approximately115 kd, and earlier studies of genetic polymorphisms indicate that they are closely linked. C8is composed of three chains (~, 64 kd; r, 64 kd; ~, 22 kd; the ~ and ~ are disulphidebonded);all are encodedon chromosome 1 (155). Formationof the C5b-8complexand its role as catalyst in the polymerizationof C9(single chain of 71 kd) to give MACs with a composition C(Sb-8)C9n(where n = 1-18) is covered in recent reviews(3). Oneemergingfeature of classical complement lysis, however, is that the ring-like lesion formedwith high ratios of C9to C5b-8 is not alwaysa prerequisite for cell lysis; complexescarrying low numbersof C9 can also efficiently cause lysis via the productionof smaller hydrophobic membranechannels. C7and the a and fl chains of C8and C9have all been recently cloned from humanliver eDNA libraries (3, 156, 157), thus allowing comparison of their derived aminoacid sequences. These show21%-26% identity on alignmentwith each other. All four chains have a large internal domain almost free of cysteine residues, an N-terminalcysteine-rich domainhomologousto the LDLreceptor repeat type A, and a C-terminal domain homologousto LDLreceptor type B epidermal growth factor type (EGFlike) sequences. Studies on the structure of the humanC9 geneindicate that the coding region is encodedby 12 exons covering at least 80 kb of DNA (158). Unexpectedly,the cysteine-rich sequences are not found encodedby discrete exons as they are in other homologous proteins such as the LDLreceptor and urokinase, and this mayindicate that the C9 geneis moreclosely related to an ancestral cysteine-rich sequencethan to these proteins. Control of C5b-9is mediatedby the S-protein for whicheDNA clones have been isolated from a humanliver eDNA expression library (159). This study has shownthat S-protein is identical to plasmavitronectin, a memberof the family of substrate adhesionmoleculessuch as fibronectin and laminin.

Annual Reviews 188

CAMPBELL,LAW, REID & SIM

Annu. Rev. Immunol. 1988.6:161-195. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Perforin (71 kd), the pore-forming protein present in the granules cytotoxic T lymphocytes, shows structural and functional similarity to the complement component C9, especially in its ability to form tabular membrane lesions (160). A eDNAclone coding for the C-terminal portion of mouse perforin predicts an amino acid sequence that is 27% homologous with the equivalent portion of human C9. This emphasizes the structural and functional similarities between perforin and C9, C8~, CSfl, and C7 and suggests that the terminal complement components are members of a widespread family of membrane-disrupting proteins.

Literature Cited I. Miiller-Eberhard, H. J., Miescher,P. A., eds. 1985. Complement.Heidelberg: Springer-Verlag. 2. Schumaker,V. N., Zavodsky,P., Poon, P. H. 1987.Activationof the first component complement.Ann. Rev. lmmunol. of 5:2t-42 3. Miiller-Eberhard, H. J. 1986. The membraneattack complex of complement. Ann. Rev. lmmunol.4:503-28 4. Reid, K.B.M.1985. Molecularcloning and characterization of the cDNA and gene coding for the B-chain of Clq of the humancomplementsystem. Biochem. J. 231:729-35 5. Sellar, G. C., Goundis,D., McAdam, R. A., Solomon,E., Reid, K. B. M. 1987. Cloningand chromosomal localisation of humanClq A-chain. Identification of moleculardefect in a Clq deficient patient. Complement 4:225 6. Tenner, A. J., Volkin, D. B. 1986. Complement subcomponentC lq secreted by cultured humanmonocyteshas a subunitstructure identical with that of serumClq. Biochem.J. 233:451-58 7. Rabs, V., Martin, H., Hitschold, T., Golan, M. D., Heinz, H. P., Loos, M. 1986.Isolation andcharacterisation of macrophage-derived Clq and its similarities to serumClq. Eur. J. lmmunol. 16:118346 8..Jouruet, A., Tosi, M. 1986. Cloning and sequencing of full-length eDNA encoding the precursor of human complementcomponentC1 r. Biochem. J. 240:783-87 9. Tosi, M., Duponche, C., Meo, T., Julier, C. 1987. CompleteeDNAsequence of humanCls and close physical linkage of the homologous genes Cls and Cir. Biochemistry.In press 10. Arlaud, G. J., Colomb, M. G., Gagnon,J. 1987. A functional model

of the humanC1 complex. Immunology Today8:106--10 11. Alper, C. A. 1981. Complementand the MHC.In The Role of the Major Histocompatibility Complexin Immunobiology, ed. M. D. Dorf, pp. 173220. NewYork: Garland 12. Mauff, G., Alper, C. A., Awdeh,Z., Batchelor, J. R., Bertrams,J., BruunPetersen, G., Dawkins,R. L., Demant, P., Edwards, J., Grosse-Wilde, G., Hauptmann, G., Klouda, P., Lamm, L., Mollenhauer, E., Nerl, C., Olaissen, B., O’Neill, G., Rittner, C., Roos,M. H,, Skanes,V., Teisberg,P., Wells,L. 1983. Statement on the nomenclature of humanCAallotypes. Immunobiolotty 164:184-91 13. Weitkamp,L. R., Lamm,L. U. 1982. Reportof the committeeon the genetic constitution of chromosome 6. Cyto#enet. Cell Genet.32:130-43 14. Chaplin, D. D. 1985. Molecularorganisation and in vitro expression of murineclass III genes. Immunol.Rev. 87:61~80 15. Campbell, R. D., Carroll, M. C., Porter, R. R. 1986. Themoleculargenetics of componentsof complement. Adv. lmmunoL38:203-44 16. Carroll, M. C., Campbell, R. D., Bentley, D. R., Porter, R. R. 1984. A molecular map of the humanmajor histocompatibility complexclass III region linking complementgenes C4, C2 and Factor B. Nature 307:237-41 17. Wu,L. C., Morley, B. J., Campbell, R. D. 1987. Expression of the human complementprotein Factor B gene: Evidencefor the role of twodistinct 5’ flanking elements. Cell 48:331-42 18. Carroll, M. C., Campbell, R. D., Porter, R. R. 1985. Themappingof 21hydroxylase genes adjacent to tom-

Annual Reviews

Annu. Rev. Immunol. 1988.6:161-195. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

COMPLEMENT SYSTEMGENES plement componentC4 genes in HLA, the major histocompatibility complex in man.Proc. Natl. Acad.Sci. USA82: 521-25 19. White,P. C., Grossberger,D., Onufer, B. J., Chaplin, D. D., New,M. I., Dupont, B., Strominger, J. L. 1985. Twogenes encodingsteroid 21-hydroxylase are located near the genes encoding the fourth componentof complementin man.Proc. Natl. Acad.Sci. USA82:1089-93 20. Higashi, Y., Yoshioka, H., Yamane, M., Gotoh, O., Fujii-Kuriyama, Y. 1986. Completenucleotide sequence of two steroid 21-hydroxylase genes tandemly arranged in human chromosome:a pseudogeneand a genuine gene. Proc. Natl. Acad.Sci. USA83: 2841-45 21. Rodrigues,N. R., Dunham,I., Yu, C. Y., Carroll, M. C., Porter, R. R., Campbell, R. D. 1987. Molecular characterisation of the HLA-linked steroid 21-hydroxylaseB gene froman individual with Congenital Adrenal Hyperplasia. EMBO J. 6:1653-61 22. White, P. C., New,M. I., Dupont,B. 1986. Structure of humansteroid 21hydroxylasegenes. Proc. Natl. Acad. Sci. USA83:5111-15 23. Chaplin,D. D., Galbraith, U J., Seidman,J. G., White,P. C., Parker, K. L. 1986. Nucleotidesequenceanalysis of murine 21-hydroxylase genes: mutations affecting geneexpression.Proc. Natl. Acad.Sci. U.S.A. 83:9601~15 24. Anand,R. 1986. Pulsedfield gel electrophoresis: a technique for fractionating large DNA molecules.Trends Genet. 2:278-83 25. Ragoussis,J., vander Bliek, A., Trowsdale, J., Ziegler, A. 1986. Mappingof HLAgenes using pulsed-field gradient electrophoresis. FEBSLett. 204:1-4 26. Lawranee,S. K., Smith, C. L., Srivastava, R., Cantor,C. R., Weissman, S. M. 1987. Megabase-scale mappingof the HLAgene complexby pulsed field gel electrophoresis. Science235:138790 27. Dunham, I., Sargent, C. A., Trowsdale, J. Campbell, R. D. 1987. Molecular map of the human major histocompatibility complexby pulsed field gel electrophoresis. Proc. Natl. Acad. Sci. USA.In press 28. Spies, T., Morton,C. C., Nedospasov, S. A., Fiers, W.,Pious, D., Strominger, J. L. 1986.Genesfor the tumornecrosis factors ~ and/3are linked to the human majorhistocompatibi/itycomplex,l~roc. Natl. Acad. Sci. USA83:8699-8702

189

29. Miiller, U., Stephan,D., Phillippsen, P., Steinmetz, M. 1987. Orientation and molecular map position of the complementgenes in the mouse MHC. EMBOJ. 6:369-73 30. Bentley, D. R., Campbell,R. D. 1986. C2 and Factor B: structure and genetics. Biochem.Soc. Syrup. 51:7-18 31. Campbell, R. D. 1987. Moleculargenetics of C2 and Factor B. Br. Med. Bull. 43:37-49 32. Agnello, V. 1978. Complement deficiency states. Medicine57:1-23 33. Cole, F. S., Whitehead,A. S., Auerbach, H. S., Lint, T., Zeity, H. J., Kilbridge, P., Colten, H. R. 1985. The molecularbasis for genetic deficiency of the second component of human complement.NewEn#l. J. Med. 313: 11-16 34. Falus, A., Wakeland,E. K., MeConnell, T. J., Gitlin, J., Whitehead, A.S., Colten, H. R. 1987. DNApolymorphismof MHC III genes in inbred and wild mouse strains. Immunogenetics 25:290-98 35. Campbell, R. D., Morley, B. J., Sargent, C. A., Janjua, N. J. 1985. Molecularbasis for allelic variation at the Factor B locus. Complement 2: 1415 36. Colten, H. R., Dowton,S. B. 1986. Regulation of complementgene expression. Biochem.Soc. Syrup. 51: 3746 37. Perlmutter, D. H., Goldberger, G., Dinarello, C. A., Mizei, S. B., Colten, H. R. 1986. Regulation of class III majorhistocompatibility complexgene productsby interleukin-1. Science32: 850-52 38. Strunk, R., Cole, S., Perlmutter, D., Colten, H. 1985.~-interferon increases expression of class III complement genes C2 and Factor B in humanmonoeytes andin murinefibroblasts transfected with humanC2 and factor B genes. J. Biol. Chem.260:15280-285 39. Friedman, R. L., Stark, G. R. 1985. Alpha-interferon-induced transcription of HLAand metallothionein genes containing homologous upstream sequences. Nature 314:637-39 40. Wu,L. C., Campbell,R. D. 1987. In preparation 41. Rogers, B. L., Saunders, B. F. 1986. Transcriptionalenhancersplay a major role in geneexpression.Bioessays4: 6263 42. Belt, K. T., Carroll, M.C., Porter, R. R. 1984. The structural basis of the multiple forms of humancomplement componentC4. Cell 36:907-14

Annual Reviews

Annu. Rev. Immunol. 1988.6:161-195. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

190

CAMPBELL,LAW, REID & SIM

43. Belt, K. T., Yu,C. Y., Carroll, M.C., Porter, R. R. 1985. Polymorphismof human complement C4. Immunogenetics 21:173-80 44. Yu, C. Y., Belt, K. T., Giles, C. M., Campbell,R. D., Porter, R. R. 1986. Structural basis of the polymorphism of human complement components C4Aand C4B:genesize, reactivity and antigenicity. EMBO J. 5:2873-81 45. Dodds,A. W., Law,S. K. A., Porter, R. R. 1986. Thepurification andproperties of someless common allotypes of the fourth component of human complement.Immunogenetics24: 27985 46. Giles, C, M. 1987. Three Chidodeterminants detected on the B5Rg+ of humanC4: their expression in Chtyped donors and families. Human Immunol, 18:111-122 47. Schneider,P. M., Carroll, M.C., Alper, C. A., Rittner, C., Whitehead,A. S., Yunis,E. J., Colten, H. R. 1986. Polymorphism of human complement C4 and steroid 21-hydroxylase gener Restriction fragment length polymorphisms revealing structural deletions, homoduplications,and size variants. J. Clin. Invest. 78:650-57 48. Palsdottir, A., Fossdal, R., Arnason, A., Edwards,J. H., Jensson, O. 1987. Heterogeneityof humanC4 gene size. lmmunogenetics25:299-304 49. Hauptmann, G., Goetz, J., UringLambert, B., Grosshans, E. 1986. C4 deficiency. Prog.Allergy. 39:232-49 50. Raum,D., Awdeh,S. L., Anderson, J., Strong,L., Granados,J., Pevan,L., Giblett, E., Yunis,E. J., Alper,C. A. 1984. HumanC4 haplotypes with duplicated C4Aor C4B.Am. J. Hum. Genet. 36:72-79 51. Carroll, M.C., Belt, K. T., Palsdottir, A., Porter, R. R. 1984, Structure and organisation of C4genes. Phil. Trans. R. Soc. Lond. B306:379-88 52. Schendel,D. J., O’Neill, G., Wank,R. 1984. MHC-linkedClass III genes. Analysesof C4 gene frequencies, complotypes andassociations with distinct HLAhaplotypes in GermanCaucasians. Immunogenetics 20:23-31 53. Partanen, J., Koskimies, S. 1986. HumanMHC class III genes, Bf and C4. Polymorphism,complotypes and association with MHC Class I genes in the Finnish population. HumanHered. 36:269-75 54. Fielder, A. H. L., Walport,M.J., Batehelor, J. R., Rynes,R. I., Black, C. M., Dodi,I. P., Hughes,G. R. V. 1983. Family study of the major histo-

compatibilitycomplexin patients with systemic lupus erythematosus.Importance of null alleles of C4AandC4Bin determiningdisease susceptibility. Br. Med. J. 286:425-28 55. Carroll, M.C., Palsdottir, A., Belt, K. T., Porter, R. R. 1985. Deletion of complement C4 and steroid 21-hydroxylase genesin the HLAclass III region. EMBOJ. 4:2547-52 56. Carroll, M.C., Alper,C. A. 1987.Polymorphismand molecular genetics of humanC4. Br. Med.Bull. 43:50-65 57. Yu, C. Y., Campbell, R. D. 1987. Definitive RFLPsto distinguish between the human complement C4A/ C4B isotypes and the major Rodgers/Chido determinants. Application to the study of C4 null alleles. lmmunogenetics25:383-390 58. Palsdottir, A., Arnason,A., Fossdal, R., Jensson, O. 1987. Geneorganisation ofhaplotypesexpressingtwo different C4Aallotypes. Hum.Genet. 76: 220-224 59. Ogata, R. T., Sepich, D. S. 1985. Murinesex-limited protein: complete eDNAsequence and comparison with murine fourth complement component. J. lmmunol.135:4239~4 60. Nonaka, M., Nakayama,K., Yeul, Y. D., Takahashi, M. 1986. Complete nucleotide and derived amino acid sequencesof sex-limited protein (Sip), non-functional isotype of the fourth component of136:2989-93 mousecomplement(C4). J. Immunol. 61. Sepich, D. S., Rosa, P. A., Ogata, R. T. 1987. cDNA sequenceof a novel sexlimited protein (Sip) frommiceconstitutive for Sip expression.J. Biol. Chem. 262:4935-38 62. Nonaka,M., Kimura,H., Yuel, Y. D., Yokayama, S., Nakayama,K., Takahashi, M. 1986.Identification of the 5’-flanking regulatory region responsible for the difference in transcriptional control between mouse complementC4and Slp genes. Proc. Natl. Acad. Sci. U.S.A. 83:7883-87 63. Nakayama,K., Nonaka, M., Yokoyama, S., Yeul,Y. D., Pattanakitsakul, S.-N., Takahashi, M. 1987. Recombination of two homologous MHC class III genes of the mouse(C4 and Slp) that accountsfor the loss of testosterone dependenceof sex-limited protein expression. J. lmmunol.138: 620-27 64. Wu,L. C., Yu, C. Y., Morley,B. J., Campbell, R. D. 1987. Regulation of expression of the complementcomponent genes encoded in the major

Annual Reviews

Annu. Rev. Immunol. 1988.6:161-195. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

COMPLEMENT SYSTEMGENES histocompatibility complex. Complement 4:240-41 65. de Bruijn, M. H. L., Fey, G, H. 1985. Human complement component C3: eDNAcoding sequence and derived primary structure. Proc. Natl. Acad. Sci. USA82:708-12 66. Lundwall,A., Wetsel, R. A., Domdey, H., Tack,B. F., Fey, G.H. 1984.Structure ofmurine complementcomponent C3: I. Nucleotide sequenceof cloned complementary and genomic DNA codingfor the fl chain. J. Biol. Chem. 259:13851-56 67. Wetsel,R. A., Lundwall,A., Davidson, F., Gibson,T., Tack,B. F., Fey, G. H. 1984. Structure of murinecomplement component C3: II. Nucleotidesequence of cloned complementaryDNAcoding for the ~t chain. J. Biol. Chem.259: 13857-62 68. Nonaka, M., Nakayama,K, Yuel, Y. D., Takahashi, M. 1985. Complete nueleotide and derived amino acid sequencesof the fourth complement of mousecomplement(C4): evolutionary aspects. J. Biol. Chem.260:10936-43 69. Sepich, D. S., Noonan,D. J., Ogata, R. T. 1985. CompleteeDNAsequence of the fourth componentof murine complement.Proc. Natl. Acad. Sci. USA 82:5895-99 70. Wetsel, R. A., Ogata,R. T., Tack, B. F. 1987.Primarystructure of the fifth component of murine complement. Biochemistry 26:737-43 71. Lundwall,A. B., Wetsel, R. A., Kristensen, T., Whitehead,A. S., Woods, D. E., Ogden,R. C., Colten, H. R., Tack, B. F. 1985. Isolation and sequence analysis of a eDNAclone encoding the fifth complementcomponent. J. Biol. Chem.260:2108-12 72. Kusano,M., Choi, N.-H., Tomita, M., Yamamoto, K., Migita, S., Sekeiya,T., Nishimura, S. 1986. Nucleotide sequence of DNAand derived aminoacid sequenceof rabbit complementcomponent C3 ~-chain. Immunol. Invest. 15:365-78 73. Whitehead,A. S., Solomon,E., Chambers, S., Bodmer, W.F., Povey,S., Fey, G. 1982. Assignmentof the structural gene for the third componentof human complementto chromosome19. Proc. Natl. Acad. Sci. USA79:5021-25 74. da Silva, F. P., Hoecker,G. F., Day, N. K., Vienne,K., Rubinstein,P. 1978. Murine complement component 3: Genetic variation and linkage to H-2. Proc. Natl. Acad.Sci. USA75:963-65 75. Natsuume-Sakai,S., Hayakawa,J. I., Takahashi, M. 1978. Genetic poly-

191

morphismof murine C3 controlled by a single co-dominantlocus on chromosome17. J. Immunol.121:491-98 76. Wiebauer, K., Domdey,H., Diggelmann,H. Fey, G. 1982. Isolation and analysis of genomicDNA clones encoding the third componentof mouse complement.Proc. Natl. Acad. Sci. USA79:7077-81 77. D’Eustachio, P., Kristensen, T., Wetsel, R. A., Riblet, R., Taylor, B. A., Tack, B. F. 1986. Chromosomal location of the genes encoding complement componentC5 and factor H in the mouse.J. Immunol.137: 399095 78. Law, S. K. A., Gagnon,J. 1985. The primary structure of the fourth component of humancomplement(C4)-C-terminal peptides. Biosci. Rep. 5: 913-21 79. Gigli, I., vonZabern,I., Porter, R. R. 1977.Theisolation andstructure of C4, the fourth componentof humancomplement. Biochem.J. 165:439-46 80. Karp,D. R., Parker, K. L., Shrettier, D. C., Slaughter,C., Capra,J. D. 1982. Aminoacid sequence homologies and glycosylation differences betweenthe fourth componentof murine complementandsex-limitedprotein. Proc.Natl. Acad. Sci. USA79:6347-49 81. Tack, B. F., Morris, S. C., Prahl, J. W. 1979. Third componentof human complement: structural analysis of the polypeptidechains of C3 andC3b. Biochemistry 18:1497-1503 82. Fernandez,H. N., Hugli, T. E. 1978. primarystructural analysisof the polypeptide portion of humanC5aanaphylatoxin: Polypeptidesequencedetermination and assignment of the oligosaccharide site in C5a. J. Biol. Chem.attachment 253:6955-64 83. Catterall, C. F., Lyons,A., Sim,R. B., Day,A. J., Harris, T. J. R. 1987,Characterization of the primaryaminoacid sequence of humancomplementcontrol protein Factor I froman analysis ofcDNA clones. Biochem.J. 242: 84056 84. Yuan, J. M., Hsiung, L. M., Gagnon, J. 1986. CNBrcleavage of the light chain of humancomplementfactor I and alignmentof the fragments. Biochem. J. 233:339-45 85. Tack,B. F. 1985.Thefl-Cys-),-Gluthiolester bondin humanC3, C4,and ct 2macroglobulin.In Complement,ed. H. J. M/Jller-Eberhard, P. A. Miescher, pp. 49-72. NewYork: Springer-Verlag 86. Sottrup-Jensen, L. 1987. ~t2-macroglobulinand related thiol ester plasma

Annual Reviews

Annu. Rev. Immunol. 1988.6:161-195. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

192

CAMPBELL,LAW, REID & SIM

proteins. In The PlasmaProteins, Vol. V, pp. 191-291. NewYork: Academic 87. Iijima, M., Tobe, T., Sakamoto,T., Tomita, M.1984. Biosynthesis of the internal thioester bondof the third componentof complement.J. Biochem. 96:1539-46 88. Thomas, M. L., Tack, B. F. 1983. Identification andalignmentof a thiol ester site in the third componentof guinea pig complement.Biochemistry 22:942-47 89. Sottrup-Jensen, L., Stepanik, T. M., Wierbicki, D. M., Jones, C. M., Lonblad, P. B., Kristensen,T., Mortensen, S. B., Petersen, T. E., Magnusson, S. 1983. The primary structure of macroglobulin and localization of a factor XIII cross-linkingsite. Ann.N.Y. Acad. Sci. 421:41-60 90. Gehring, M. R., Shiels, B. R., Northemann,W.,de Bruijn, M.H. L., Kan, C-C., Chain,A. C., Noonan,D. J., Fey, G. H. 1987. Sequenceof rat liver macroglobulinand acute phase control of its messengerRNA.J. BioL Chem. 262:446-54 91. Law,S. K., Levine, R. P. 1977.Interaction betweenthe third complement protein and cell surface macromolecules. Proc. NatLAcad.Sci. USA74: 2701~)5 92. Law,S. K. A., Dodds,A. W., Porter, R. R. 1984. A comparisonof the properties of the twoclasses, C4Aand C4B, of the humancomplementcomponent C4. EMBO J. 3:1819-23 93. Isenman,D. E., Young,J. R. 1984. The molecularbasis for the difference in immune hemolysisactivity of the Chido and Rodgers isotype of humancomplement component CA. J. lmmunoL 132:3019--27 94. Dodds, A. W., Law, S. K. A. 1987. Structural basis of the bindingspeci, ficity of the thioester containingproteins, C4, C3 and ~t~-macroglobulin. ComplementSubmitted 95. Whaley,K. 1980. Biosynthesis of the complementcomponentsand the regulatory proteins of the alternative pathwayby humanperipheral blood monocytes. J. Exp. Med.151:501-16 96. Goldberger,G., Bruns,G. A. P., Pits, M., Edge, M. D., Kwiatkowski,D. J. 1987. Humancomplement factor I: analysis of cDNA-derivedprimary structure. Biochemistry262:10065-71 97. Stanley, K. K., Page, M., Campbell,A. K., Luzio, J. P. 1986. Mechanism for the insertion of complement component C9 into23:451-58 a target membrane. Mol. Immunol.

98. Doolittle, R. F. 1985.Thegenealogyof somerecently-evolvedvertebrate proteins. TrendsBiochem.Sci. 10:233-37 99. Nishimukai, H., Tamaki, Y. 1986. 1 typing by Agarose Gel Isoelectric Focussing. HumanHered. 36:195-97 I00. Rasmussen, J. M., Teisner, B., Brandslund,I., Svehag,S-E. 1986. A family with complement factor I deficiency. Scand. J. lmmunol.23:71115 101. Holers, V. M., Cole, J. L., Lublin, D, M., Seya, T., Atkinson, J. P. 1985. HumanC3b- and C4b-regulatory proteins: A newmulti-genefamily. Immunol. Today6:18.8-92 102. Reid, K. B. M., Bentley, D. R., Campbell,R. D., Chung,L. P., Sim,R. B., Kristensen, T., Tack, B. F. 1986. Complementsystem proteins which interact with C3bor C4b. lmmunol. Today 7:230-33 103. Kristensen,T., D’Eustachio,P., Ogata, R. T., Chung,L. P., Reid, K. B. M., Tack, B. F. 1987. The superfamily of C3b/C4b-binding proteins. Fed. Proc. 46:2463459 104. Klickstein, L. B., Wong,W.W.,Smith, J. A., Weis, J. H., Wilson, J. G., Fearon, D. T. 1987. HumanC3b/C4b receptor (CR1):Demonstrationof long homologous repeating domains. J. Exp. Med. 165:1095-1112 105. Rey-Campos, J., Rubinstein, P., Rodriguez de Cordoba, S. 1987. Mapping of DAFto the RCAgene cluster in humans. Complement4:217 106. RodriguezdeCordoba,S., Rubinstein, P. 1987.Newalleles of C4BindingProtein and Factor H. Immuno#enetics 25: 267-68 107. Rodriguez de Cordoba, S., Rubinstein, P. 1987. Quantitative variations of CR1 in human erythrocytes are controlled by genes within the RCAgene duster. J. Exp. Med. 16~: 1274-83 108. Barnum,S., Kenney,J., Kristensen,T., Noack, D., Seldon, M., D’Eustachio, P., Chaplin, D., Tack, B. 1987. Chromosomallocation and structure of the mouseC4bpgene. Complement4:131 109. Carroll, M.C., Alicot, E. A., Katzman, P., Klickstein, L. B., Fearon, D. T. 1987. Organisationof the genes encoding CR1,CR2, DAFand C4bpin the RCAlocus on human chromosome1. Complement4:141 110. Ripoche,J., Day,A. J., Harris, T. J. R., Sim, R. B. 1988. Completeamino acid sequence of humancomplement factor H. Biochem.J. 249: In press 111. Kristensen, T., Tack, B. F. 1986.

Annual Reviews

Annu. Rev. Immunol. 1988.6:161-195. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

COMPLEMENT SYSTEMGENES Murineprotein H is comprisedof 20 repeating units, 61 amino acids in length. Proc. Natl. Acad.Sci. USA83: 3963-67 112. Alsenz, J., Schulz, T. F., Lambris,J. D., Sim, R. B., Dierich, M. P. 1985. Structural and functional analysis of the complementcomponentfactor H with the use of different enzymesand monoclonal antibodies to factor H. Biochem.J. 232:841-50 113. Schwaeble,W.,Schulz, T. F., Zwirner, J., Dierich, M.P., Weiss,E. H. 1987. Complement factor H: expression of an additional truncated mRNA and corresponding protein in humanliver. Complement4:224 114. Vik, D. P., Keeney,J. B., Bronson,S. Westlund,B., Kristensen,T., Chaplin, D. D., Tack, B. F. 1987. Analysis of the murine factor H gene and related DNA.Complement4:235 115. McAleer,M. A., Hauptmann,G., Brai, M., Misiano, G., Sim, R. B. 1987. Restriction fragmentlength studies for factor H. Complement 4:191 116. Lopez-Larrea, C., Dieguez, M. A., Enguix, A., Dominguez, O., Gomez,E. 1987.A familial deficiency of complementfactor H. Biochem.Soc. Trans. 15. In press 117. Brai, M., Misiano, G., Hauptmann, G. 1985. Hereditarydeficiency of complement factor H. Complement2: 1213 118. Perkins, S. J., Chung,L. P., Reid, K. B. M. 1986. Unusualultrastructure of complement-componentC4b-binding protein of humancomplementby synchrotron x-ray scattering and hydrodynamicanalysis. Biochem.J. 233: 799-807 119. Chung,L. P., Bentley, D. R., Reid, K. B. M. 1985. Molecular cloning and characterisation of the cDNA coding for C4bbinding protein. Biochem.J. 230:133-41 120. Lintin, S. J., Lewin,A., Reid,K. B. M. 1987. Studies on the structure of the human C4b-binding protein gene. Complement4:186 121. Kristensen,T., Ogata,R. T., Chung,L. P., Reid, K. B. M., Tack, B. F. 1987. Cloning and eDNAsequence of mouse complementC4bp. Biochemistry. 26: 4668-74 122. Lintin, S. J., Reid,K. B. M.1986.Studies on the structure of the humanC4bbinding protein gene. FEBSLetts. 204: 77-81 123. Sim, R. B., Malhotra,V., Day, A. J., Erdei, A.1987.Structureandspecificity of complementreceptors. Immunol.

193

Left. 14:183-90 124. Hourcade,D., Holers, V. M., Miesner, D. R., Atkinson,J. P. 1987. Alternate processing during CR1transcription. Complement4:172 125. Holers, V. M., Chaplin, D. D., Leykam, J. F., Gruner, B. A., Kumar, V., Atkinson, J. P. 1987. HumanCR1 mRNA polymorphismcorrelates with the CR1allelic molecularweight polymorphism.Proc. Natl. Acad.Sci. USA 84:2459-63 126. Klickstein, L. B., Rabson, L. D., Wong,W. W., Smith, J. A., Fearon, D. T. 1987. CR15’ eDNAsequences contain a fourth LHRand identify a new B cell-specific mRNA.Complement 4:180 127. Wilson, J. G., Murphy,E. E., Wong, W.W., Klickstein, L. B., Weis,J. H., Fearon, D. T. 1986. Identification of a restriction fragment length polymorphism by a CR1 eDNAthat correlates with the number of CR1on erythrocytes. J. Exp. Med.164:50-62 128. Weis,J. J., Fearon,D. T., Klickstein, L. B., Wong,W.W., Richards, S. A., deBruynKops,A., Smith, J. A., Weis, J. H. 1986. identification of a partial eDNAclone for the C3d/Epstein Barr Virus receptor of humanB lymphocytes. Proc. Natl. Acad.Sci. USA83: 563942 129. Weis,J. J., Toothaker,L. E., Burrow, S. R., Weis,J. H., Fearon,D. T. 1987. HumanCR2 is comprised of linked groups of SCRs.Complement4:258 130. Weis,J. H., AegerteroShaw, M., Cole, J. L., Tantravahi,R. V., Miller, M.D., Kurtz,C. B., Diehl, A. D., Weis,J. J. 1987. Analysis of murine complement receptor gene family. Complement4: 238 131. Aegerter-Shaw, M., Cole, J. L., Klickstein, L. B., Wong, W. W., Fearon, D. T., Lalley, P. A., Weis,J. H. 1987. Expansionof the complement receptor genefamily. J. Immunol.138: 3488-94 132. Caras, I. W.,Davitz, M.A., Rhee,L., Weddell, G., Martin, D. W., Nussenzweig, V. 1987. Cloning of decayaccelerating factor suggests novel use of splicing to generate two proteins. Nature 325:545-49 133. Medof,M. E., Lublin, D. M., Holers, V. M., Ayers, D. J., Getty, R. R., Leykam,J. F., Atkinson,J. P., Tycocinski, M.L. 1987.Cloningandcharacterisation of cDNAsencoding the completesequenceof decay accelerating actor. Proc. NatLAcad.Sci. USA 84: 2007-I1

Annual Reviews

Annu. Rev. Immunol. 1988.6:161-195. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

194

CAMPBELL,LAW, REID & SIM

134. Stafford, H. A., Tykocinski, M, L., Holers, V. M., Lublin, D. M., Atkinson, J. P., Medof,M. E. 1987. Polymorphismof the DAFgene. Complement 4:227 135. Seya, T., Ballard, L., Bora, N., McNearney, T., Atkinson, J. P. 1987. membraneCofactor Protein (MCPor gp45-70). Complement4:225 136. Sim,R. B., Malhotra,V., Ripoche,J. Day, A. J., Micklem,K. J., Sim, E. 1986. Complement receptors and related complement control proteins. Biochem. Soc. Symp. 51:83-96 137. Ross, G. D. 1986. Opsonization and membranecomplementreceptors. In Immunobiology of the Complement System: An Introduction for Researchand Clinical Medicine,ed. G. D. Ross, pp. 87-114. Orlando, Florida: Academic 138. Wright,S. D., Silverstein, S. C. 1982. Tumor-promoting phorbol esters stimulate C3b and C3b’ receptormediated phagocytosis in cultured humanmonocytes. J. Exp. Med. 156: 114944 139. Wright,S. D., Silverstein, S. C. 1983. Receptors for C3b and C3bi promote phagocytosis but not the release of toxic oxygen from humanphagocytes. J. Exp. Med.158:2016-23 140. Sanchez-Madrid, F., Nagy,J. A,, Robbins, E., Simon,P., Springer, T. A. 1983. A human leukocyte differentiation antigenfamilywithdistinct subunits and a common/~-subunit: the lymphocytefunction-associated antigen (LFA-1), the C3bi complement receptor (OKM1/Max-1), and the p150,95 molecule. J. Exp. Med. 158: 1785-1803 141. Micklem, K. J., Sim, R. B. 1985. Isolation of complement-fragmentiC3b-binding proteins by affinity chromatography:the identification of p150,95 as an iC3b-binding protein. Biochem.J. 231:233-36 142, Malhotra, ¥., Hogg, N., Sim, R. B. 1986. Ligand binding by the p150,95 antigen of U937monocyticcells: properties in commonwith complement receptor type 3 (CR3).Eur. J. Immunol. 16:1117-23 143. Keizer, G. D., Borst, J., Visser, W., Schwarting,R. de Vries, J. E., Figdor, C. G. 1987. Membraneglycoprotein p150,95 of humancytotoxic T cell clones is involved in conjugate formationwith target cells. J. Immunol. 138:3130-36 144. Springer, T. A., Dustin, M.L., Kishimoto, T. K., Marlin, S. D. 1987. The lymphocytefunction-associated LFA-1,

CD2,and LFA-3molecules: cell adhesion receptors of the immunesystem. Ann. Rev. Immunol.5:223-52 145. Law,S. K. A., Gagnon,J., Hildreth, J. E. K., Wells,C. E., Willis, A. C., Wong, A.J. 1987.Theprimarystructure of the fl-subunit of the cell surface adhesion glycoproteins LFA- 1, CR3 and p150,95 and its relationship to the fibronectin receptor. EMBO J. 6: 91519 146. Kishimoto,T. K., O’Connor,K., Lee, A., Roberts, T. M., Springer, T. A. 1987.Cloningof the fl subunit of the leukocyteadhesionproteins: homology to an extracellular matrix receptor defines a novelsupergenefamily. Cell 48:681-90 147. Wells, C. E., Law,S. K. A. 1987. RFLP of the fl-subunit of the cell surface adhesion glycoproteins. Complement 4:238 148. Marlin, S. D., Morton,C. C., Anderson, D. C., Springer,T. A. 1986.LFA-1 immunodeficiency disease--Definition of the genetic defect and chromosomal mappingof ct and fl subunits of the lymphocytefunction-associated antigen 1 (LFA-1)bycomplementation hybrid cells. J. Exp. Med.164:85547 149. Dana, N., Clayton, L. K., Tennen, D. G., Pierce, M. W., Lachmann,P. J., Law,S. A., Arnaout,M. A. 1987. Leukocytes from four patients with complete or partial Leu-CAM deficiency contain the common/3-subunit precursor and fl-subunit messengerRNA. J. Clin. Invest. 79:1010-15 150. Kishimoto, T. K., Hollander, N., Roberts, T. M., Anderson, D. C., Springer, T. A. 1987. Heterogenous mutationsin the fl-subunit common to the LFA-1,Mac-l, and p150,95 glycoproteins cause leukocyte adhesion deficiency. Cell. 50:193-202 151. Tamkun, J. W., DeSimone, D. W., Fonda,D., Patel, R. S., Buck,C., Horwitz, A., Hynes,R. O. 1986. Structure of integrin, a glycoproteininvolvedin the transmembrane linkage between fibronectin and actin. Cell 46:271-82 152. Fitzgerald,L. A., Steiner, B., Rall, S. C. Jr., Lo, S-S., Phillips, D. R. 1987. Protein sequenceof endothelial glycoprotein IIIa derived from a cDNA clone: Identity with platelet glycoproteinBioL IIIaChem. and similarity to "integrin". J. 262:3936-39 153. Miller, L. J., Wiebe,M,, Springer, T. A. 1987. Purification and~-subunit Nterminal sequences of humanMac-1 and p150,95 leukocyte adhesion proteins. J. Immunol.138:2381-83

Annual Reviews

Annu. Rev. Immunol. 1988.6:161-195. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

COMPLEMENT SYSTEMGENES

195

154. Hynes,R. O. 1987. Integrins: A family cysteine-rich domainsof C9. Compleof cell surfacereceptors. Cell 48: 549ment 4:189 54 159. Jenne, D., Stanley, K. K. 1985. Mo155. Rogde,S., Olaisen,B., Gedde-Dahl, T., lecular cloning of S-protein, a link Teisberg, P. 1986. The C8Aand C8B betweencomplement,coagulation and loci are closely linked on Chromosome cell-substrate adhesion. EMBO J. 4: 1. Ann. Hum.Genet. 50:139-44 3153-57 156. DiScipio, R. G., Chakravarti, D. N., 160. Young,J. D., Cohn, Z. A., Podack, Miiller-Eberhard, H. J., Fey, G. H. E. R. 1986. The ninth componentof 1987. The structure of human C7. complementand the pore-formingproComplement4:150-51 tein (perforin1) fromcytotoxicT cells. 157. HowardO. M.Z., Rao, A. G., Sodetz, Science 233:184-90 J. M. 1987. eDNAand derived amino 161. Hardy,D. A., Bell, J. I., Long,E. O., acid sequenceof the/~-subunit of C8: Lindsten, T., McDevitt,H. O. 1986. identification of a close structural and Mappingof the class II region of the ancestral relationship to the ~t-subunit humanmajor histocompatibility comand C9. Biochemistry.In press plexby pulsed-fieldgel electrophoresis. 158. Marazziti,D., Eggersten,G., Stanley, Nature 323:453-55 K. K., Fey, G. 1987. Evolution of the

Annu. Rev. Immunol. 1988.6:161-195. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Annu. Rev. Immunol. 1988.6:161-195. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Annual Reviews www.annualreviews.org/aronline Ann.Rev. lmmunol.1988. 6 : 197-218 Copyright©1988by AnnualReviewsInc. All rights reserved

Annu. Rev. Immunol. 1988.6:197-218. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

NORMAL, AUTOIMMUNE, AND MALIGNANTCD5 + B CELLS: THE LY-1 B LINEAGE? Kyoko Hayakawa and Richard

R. Hardy

Institute for CancerResearch, FoxChaseCancerCenter, Philadelphia, Pennsylvania 19111 INTRODUCTION Overthe past decade complexityin the population of B lymphocyteshas becomeincreasingly apparent. Several studies have revealed functional and physiological heterogeneity, someeven suggesting multiple lineages of B cells (1-6). However,understandingof B cell subsets has beenbased on the essential concept that B cells as a wholeare generated from a common precursor in bone marrow.In contrast, recent study of a murine B cell subpopulation, Ly-1 B, has led to reconsideration of B cell heterogeneityfrom a developmentalpoint of view. Thedeficiency of adult bone marrowin generating Ly-1 B, in particular, raised the question whetherB cells mightalready be distinct at the progenitorlevel of development. This hypothesis eventually enabled us to construct a frameworkfor understandingCD5(Ly- 1 in mouse)expressionon B cells, originally found in two distinct contexts: (a) on chronic malignantB cells; and (b) fraction of splenic B cells in the autoimmune-prone mousestrain, NZB. In this reviewwediscussavailable evidencefor understandingthese initial aspects--generation of chronic malignancyand autoantibodyspecificity-fromthe viewthat CD5expressingcells constitute a separate lineage of B cells. DISCOVERY OF CD5 EXPRESSION MALIGNANT B CELLS

ON CHRONIC

Theexp.ressionof a T cell differentiation antigenon certain B cell tumors wasfirst reported in a humanleukemiastudy in 1978(7). This expression 197 0732-0582/88/0410-0197502.00

Annual Reviews

Annu. Rev. Immunol. 1988.6:197-218. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

198

HAYAKAWA & HARDY

wasfounduniquelylimited to Ig + cells in B chronic lymphocyticleukemia (B CLL)(and B lymphocyticlymphoma) and was not detected in ÷ cells in acute B leukemia, Burkitt’s lymphoma, or other B cell proliferative disorders (8-11). Later, the antigen was determined to be Leu-1 (now knownas CD5)(9). At present, Leu-1is the most clearly defined antigen reported to be found on both normalmatureT cells and chronic malignant B cells but not on the majority of normalB cells. In fact, screeningof a panel of monoclonalantibodies knownto react with both B CLLand T cells showedthat all of these antibodiesrecognizedepitopes present on the Leu-1 molecule(12). Early work with murine B cell tumors mostly dependedon deliberate induction with carcinogensor virus. In contrast, in 1978, Lanier et al reported a high incidence of murineB-cell lymphoma in a newstrain of mouse, Bl0-H-2aH-4bp/Wys(2a4b), promoted in a novel way: hyperimmunization with the antigen sheepred blood ceils (SRBC) (13). At about the sameperiod, Slavin & Strober also reported a case of murineB-cell leukemia (BCL3that occurred spontaneously in an old BALB/cmouse, suggesting a mouseequivalent of humanB CLL(14). These lymphomas were all characterized by markedsplenomegalyand lymphnode enlargement, transplantability, and primary lack of thymus or bone marrow involvement. Analyzinga series of B cell lines (CHseries) from2a4b mice, Lanier et al foundthat Ly-1is expressedon several CHlines (15, 16). TheLy-1level varies amongCHlines; later, moresensitive analysis clearly suggestedthat almostall of the CHlines (and BCL~; 17) expressLy-1(18, 19). In support of this evidencefor the high incidenceof Ly-1expressionon late-appearing B lymphomas, Davidsonet al found that almost all cell lines derived from individual B lineage lymphomasoccurring in NFS/Nv-congenic mice do express Ly-1 (20). As with humanB CLL,Ly-1 is the only murineT cell differentiation antigen foundon such murineB cell lines. HOMOLOGY OF LEU-1

AND LY-1

Homologybetween humanLeu-1 (CD5) and mouse Ly-1 molecules had already been predicted from their distribution on several cell types and fromtheir similar size (67K)(9, 15, 21). cDNA clones have beenisolated recently for both Leu-1(22) andLy-1(23). Thesequencesof Leu-1and show63%identity along with strong (90%) homologyin their carboxyterminal regions and conservationof a cystein-rich amino-terminalregion (22, 23). Althougha protein homology search revealed no extensive similarity with any other sequencein the available data bases, Ly-1and Leu-1 are considered distant membersofthe immunoglobulingene superfamily

Annual Reviews TI-I~

LY-1 B LIN~AGE

199

becauseof predicted secondarystructure similarities in the first aminoterminal subregion and identities betweencertain conserved residues (23). Expressionof Leu-1and Ly-1moleculeson B cell lines has beenconfirmed both by immunoprecipitation(12, 15) and by Northern blot analysis (23).

Annu. Rev. Immunol. 1988.6:197-218. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

A CD5-EXPRESSING B CELL SUBPOPULATION: IMPLICATIONS FOR A B CELL LINEAGE Followingthe detection of CD5on malignantB cells, initial attempts to detect B cells with this phenotypein normaladults wereequivocal,because of the apparentpaucity of such cells (if they werepresent at all) (7-9). However,using a characteristic shared by B CLLand a subset of normal B lymphocytesof forming rosettes with mouseerythrocytes, CaligarisCappioet al discoveredthe infrequent presenceof CD5positive B cells in such a population rosetted from humanperipheral lymphoidtissues (24). Thepresenceof similar cells in micewasalso suggestedby Ledbetteret al (25). Finally, improvementsin multiparameteranalysis using the fluorescence activated cell sorter conclusively demonstratedthe presence of these cells as normalconstituents of the lymphoidcompartment. Human." Leu-1 ÷ B Cells Appear Early in Development Perhapsthe mostintriguing finding concerningthese normalcells wastheir appearancein early ontogenyand their relative predominance amongearly B cells (compared with their relative rarity in adult). In the human,Bofill et al reported (by tissue immunofluorescence analysis) that althoughLeu-1 expression was not found either on very early surface IgM- B-lineage + cells, Leu-I ÷ B cells constituted a large fraction of cells or on IgD-IgM early lymphnode after 17 wk of gestation, and furthermore, that some splenic B cells are Leu-1+ (26). Antinet al also foundthat Leu-1+ B cells werenot significant in fetal (-,~ 20 wk)liver, whereasthey constituted majorityof B cells in fetal spleen(in contrastto their rarity in adult spleen) (27). Furthermore, FACSanalysis of mononuclearcells in cord blood (from 20 wk and later) showsthat a majority (50%-95%) of B cells Leu-1÷, whereasonly 10%-20% of B cells in adult PBLare Leu-1÷ (28). Such Leu-1÷ B cells in early developmentrarely express the CALLA antigen (27). [CALLA is detected on a larger proportion of fetal hematopoietic organs, early lymphoidcells (29), and common acute leukemic cells, Burkitt’s lymphomas, but not on B CLL(30). Suchexclusive CALLA or Leu-1expressioncarries significant implications, suggestingdifferent etiologies of B cell malignancydependingon the original B cell subset affected.]

Annual Reviews 200

HAYAKAWA & HARDY

Annu. Rev. Immunol. 1988.6:197-218. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Mouse: The Ly-1 B Lineaye is SelfiMaintaining, Independent From Bone Marrow The appearance of Leu-1 B in the humanfetus suggests that there is a genetically programmedheterogeneity in the expression of Leu-1 on B cells during the course of differentiation. This was directly demonstrated recently at the progenitor level with mousecells using an in vitro culture system. From an isolated population of very early B cell progenitor(s) (B220-) in newbornliver, Ly-1 + and Ly-1 - B cell subsets were eventually generated as two distinct populations on a liver-derived stromal cell line (R. R. Hardy, T. Kishimoto, K. Hayakawa, Eur. J. Immunol. In press). It is unlikely that CD5expression occurs on already differentiated (surface Ig+) B cells since several attempts to induce Ly-1 or Leu-1 expression in various experimental conditions failed (31, 32). However,it is not formally necessary to hypothesize distinct Ly- 1 + and Ly-1- B lineages from these data relying simply on surface phenotype. Particularly in relation to malignant B cells, it remains equally possible that unknownevent(s) promote CD5expression at a high level in early cell development and that the same mechanism might also elevate CD5 expression abnormally in chronic B cell malignancy. However,a series of experiments carried out in mice provides support for the hypothesis, strongly suggesting the existence ofa Ly-1 B lineage whosenatural history is distinct from the majority of B cells in the adult. ONTOGENY AND TISSUE DISTRIBUTION:

EARLY APPEARANCE AND HIGH IN

PERITONEUM Ly-l-expressing B cells in mice were detected in spleen at a very low frequency (1%-2%) in most adult inbred mice and were termed "Ly-1 B" (33). Ly-1 B are very infrequent in spleen and peripheral blood but are curiously enriched amongthe B cells found in the peritoneal cavity (PerC) where they correspond to 10%-40%of total cells (34) (Figure In contrast, Ly-1 B are normally undetectable in bone marrow, lymph node, Peyer’s patch, and thymus(33, 34). In ontogeny, fetal liver also does not showa significant percentage of surface Ig + (or B220+) Ly-1 + cells. Possible precursors for Ly-1 B, B220+ Ly-1 + cells were only rarely detectable in bone marrow of young mice (17). However, Ly-1 B constitute higher proportion (30%) of splenic and PerC IgM+ B cells in young (1 wk) mice (33, 34), and PerC retains this high Ly-1 B percentage throughout adulthood (34). The mechanismthat maintains high levels of Ly-1 B the peritoneal cavity has not been established, although the presence of Leu-1 B cells in the humanperitoneal cavity has also been demonstrated in the early fetus (26). LY-1 B RECONSTITUTIONBY SURFACE IGM+ LY-1 B

This shift in the ratio

Annual Reviews THE LY-1

B LINEAGE

BALB~

NZB

Spleen

PerC

Spleen

Annu. Rev. Immunol. 1988.6:197-218. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

lo0 ,.~L~ IgM

~ ~o ’

100

.1

201

1

lo0 10

100

.1

1

10 100

Figure 1 Demonstration of Ly-1 B in BALB/cand NZBmice. Spleen or peritoneal cells (PerC) were simultaneously stained with Fluorescein-labded anti-IgM and phyeocyaninlabeled anti-Ly-1 antibody. Ly-1 B region is boxed.

of Ly-l-expressing B cells during developmentwas further traced to the stemand/or progenitor level by reconstitution experimentsin irradiated mice. Theexperimentsrevealed that the dichotomyseen in differentiated ÷) B cells in fact originates from changesin the stemcell(s) and/or (IgM progenitors with age (35). If B cell progenitors in adult bonemarroware the sameas the ones present in early development,then reconstituting miceshould recapitulate their normaldevelopment;that is, there should be high Ly-1B at an early time and significant numbersamongPerCB cells in the adult. In fact, liver fromnewborn micewascapableof generatingall B cell subsets, whereasin contrast, bonemarrowfrom adult mice yielded goodreconstitution of Ly-1-B cells, but few (if any) Ly-1B (35). Quite often, BMreconstituted mice lacked any detectable Ly-1 B for their remaininglifespan. A possible suppressiveeffect of adult bonemarrowon the generation of Ly-1 B is unlikely, becausea mixture of bone marrow andfetal liver still yieldedLy-1B (andthis exclusivelyfromthe fetal liver source).

Impaired production of Ly-1 B from adult bone marrowcould be explainedentirely as a qualitative changein stemor progenitorcells with age. However,the distinctive high frequency of Ly-1 B in adult PerC arguedagainst a tapering of B cell progenitor activity in bone marrow. This wasresolvedby the surprising finding of a self-reconstituting ability by Ly-1 B (35, 36). Experimentswith irradiated mice showedthat purified

Annual Reviews

Annu. Rev. Immunol. 1988.6:197-218. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

202

HAYAKAWA & HARDY

surface IgM÷ Ly-1 B (but not IgM- cells) from adult PerCcould reconstitute almost exclusively Ly-I ÷ B themselvesin both PerCand spleen and, furthermore,could maintainthis populationfor a long time (at least 6 months)(36). Actual proliferation of Ly-1 B was strongly suggested whenPerC was injected into newbornmice and these individuals were then analyzedafter they had reached adulthood(37). This characteristic is not limited to the PerCLy-1 B cells, since newbornsplenic Ig+ cells (including Ly-1B) also reconstitute exclusively the Ly-1B found in PerC (36). Takentogether, these results support the hypothesis of a Ly-1 lineage generatedearly in ontogeny,localizing in the peripheryand maintaining itself independentof the bonemarrow. NEONATAL ANTI-IGM TREATMENT Their self-renewing capacity (as distinct from other B cells) was further demonstratedby Lalor et al using the surface immunoglobulin phenotypeof Ly-1B (38). Ly-1B in mice express relatively higherlevels of surfaceIgMbut distinctively lowerlevels of IgD throughout their lifetime (33). Treatment of immunoglobulinallotype heterozygous mice with anti-IgM specific for one allotype completely abrogatesthe generationof Ly-1B of this allotype in later life, whereas other (Ly-1-) B cells appear after cessation of antibody administration. Eliminatingor blockingthe generationof Ly-1B at an early developmental stage evidentlyeliminatessuch cells completelyfor the life of the animal. + This demonstrates the importanceof neonatally generated surface IgM cells in the maintenanceof this population. Onthe other hand, anti-IgD treatment did not affect the generationof Ly-1B. GENETIC

INFLUENCE

ON THE LEVEL OF LY-1

B

Examinationof Ly- 1 B in a variety of inbred mousestrains also suggests that the generationof Ly- 1 B is independentof other B cells and reveals complexB cell differentiation profiles. Thestrains discussedhere are all consideredimmunodefective for various reasons. Whilewehave not established the significance of Ly-1B in such defects, the study of Ly-1B in these strains mayprovidea key to understandingB cell differentiation per se. Onefeature of several of these strains is autoantibodyproduction,and wereturn to this as it relates to Ly-1B in a later section. SJL Mice Asurvey of "normal"inbred mousestrains revealed genetic influences on the level of Ly-1 B in adult peritoneum,whereeach strain showsits own stable level (34). BALB/c and their H-2or immunoglobulin allotype (Igh) congenic strains have relatively high levels comparedwith others

Annual Reviews THE LY-1

B LINEAGE

203

Annu. Rev. Immunol. 1988.6:197-218. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

(C57BL/10,CBA,A.TH,129/Sv, etc.) (34), In contrast, SJL and the congenicSJAstrain both showa distinctively lower level (34, 39). These traits are apparentlycodominantlyregulated by a few genes, accordingto an analysis of micebackcrossedbetween(BALB × SJL) F1 and each parent (39, 40). However,close association or linkage to any knowngenes has not beenestablished yet and requires further investigation. Motheaten Mice The genetically defective mice, "motheaten" and "viable motheaten," carry homologous recessive allelic genes, meor mev on chromosome 6 (41, 42). Thesemicehavea short life (mean22 and61 days respectively), severe immunod¢ficiency,and autoimmunity(42). They have decreased numbers of B cells, andsurprisingly,almostall the B cells foundin mev mice(spleen, lymphnode, PerC) bear Ly-1(43). Theabsenceof normalB cells together with increased numbersof Ly-1found in mev mice supports a split lineage modelof B cell differentiation, with normalgeneration of Ly-1B (which occurs early) and deficient production of Ly-1- B cells (whichnormally appear later from the bone marrow). )~id Mice In contrast, mice bearing the X chromosome-linkedimmunodeficiency xid (2, 44, 45) haveimpairedLy-1 B generation. Analysisof Ly-1B levds in PerC showedthat CBA/N mice or male offspring of F1 mice with a CBA/N mother completely lack Ly-1 B (34). NZBmice (which normally have elevated levels of Ly-1 B, see below) congenic .for the xid gene also showthis deficiency (34). Althoughxid has a complexeffect on the developmentof the immunesystem in addition to this Ly-1 B deficit (46-48), the study of Ly-1 B mayprovide a clue to understanding the immunodeficiency characterized in this defect. Nude Mice The nudemutationdoes not result in the lack of Ly-1 B, whichimplies a relative independence of Ly-1B generationfromT cells or thymicinfluence (34). However,the environment resulting from the athymic mutation influencesthe Ly-1B level, resulting in slightly decreasedlevels of Ly-1B in BALB/cand NZBnu/nu mice comparedwith nu/÷ littermates (39; and in collaborationwith Dr. Y. Ohsugi).This suggestssomeT cell (or thymic) influence on the generation or maintenance of Ly- 1 B. ~VZB Mice Theproliferative ability of Ly-1 B in adult spleen wasstrongly manifested in NZBmice,whichshowa striking genetic effect on Ly-1B level (33, 49).

Annual Reviews

Annu. Rev. Immunol. 1988.6:197-218. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

204

HAYAKAWA & HARDY

Their level of Ly-1B is distinctively higher compared with normalstrains at 2 monthsor older (Figure 1). In particular, pauciclonalproliferation Ly-1 B occurs frequently in NZBmice, is extreme in PerC, and often occurslater in the spleen. Suchproliferation results in splenomegaly consisting almost entirely of Ly-1B, and their presenceeven in lymphnodes suggests expansioninto most lymphoidcompartments.Hyperdiploidcells, whichmayrepresent a premalignant stage (50), were found in the NZB PerC Ly-1 B population (K. Hayakawa,R. R. Hardy, unpublished). addition to such abnormalitiesfoundin older NZBmice, uniquefunctional characteristics (such as high IgMsecretion in vitro) appear early in NZB B cell development (51); this also mayrelate to Ly-1B function. However, it is not clear whetherthe genetic backgroundwhichaffects Ly-1 B so profoundlyis limited in its effects only to this subset. Nevertheless,it is clear that the proliferation of Ly-1 B (which occurs to someextent in "normal" strains with age) is strongly promotedby gene(s) in the background. TUMORGENESIS AND LY-1

(LEU-1)

Datasummarized in the previous section illustrated genetic regulation of + B cells. The following data suggest a process whereby the level of CD5 abnormalproliferation of Ly-1B takes place, particularly in later stages of development, influenced by other genetic factors. Premalignant Cells MOUSE AbnormalLy-1 B proliferation occurs in NZBand is elevated in (NZBx NZW) F 1 mice (33). Old BALB/cmice also occasionally show distinctively expandedpopulationof splenic Ly- 1 B, andthis is apparently promotedby immunizationtogether with adjuvant (K. Hayakawa,R. R. Hardy, unpublished). Ly-1 B are relatively radioresistant comparedto other B cells (34), with occasionalirradiated miceshowingexclusive Ly-1 B proliferations. Thesecases maypotentially be in a premalignantstage, finally proceedingto chronic B cell malignancy.In contrast, amonglaboratory inbred mice, the BXSB strain has normal levels of Ly-1 B but showsa markedproliferation of B cells that do not express Ly-1(and that have higher levels of IgD than do Ly-1 B) (52). This suggests that lymphoproliferationsin NZBand BXSB mice originate by different mechanisms and implies that Ly-1 B is associated with chronic malignant proliferation only whencombinedwith certain genetic backgrounds. HUMAN Genetic effects on the frequencyof humanLeu- 1 ÷ B cells (LeuB) are also suggested by association with disease. A high incidence of

Annual Reviews

Annu. Rev. Immunol. 1988.6:197-218. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

THE LY-I

B LINEAGE

205

elevated levels of Leu-1B occursamongpatients with rheumatoidarthritis (28, 53) or Sjogren syndrome(28). Sjogren’s patients are knownto an increased risk of B cell malignancy.Furthermore,Foxet al (54) have suggesteda transition of B cells fromproliferation into neoplastic transformation. ACRI(cross-reactive idiotype) present on V-KIIIbof IgM (rheumatoidfactor) froma rheumatoidarthritis patient is shared with the RF paraproteins in Waldenstrom’s macroglobulinemia and lymphoma patients. By using the antibody to this CRI, they found progressive increases in the proportion of B cells bearing the CRIin patients with Sjogrensyndrome.Althoughnot directly tested, such proliferating B cells in Sjogren’s patients could be the expandedpopulation of Ly-1 B mentioned above. Malignant Cells and Transformed Cell Lines B LYMPHOMAS IN 2a4b MICEAn extensive series of studies on the CHB lymphomashave been carried out by Haughtonet al (19). This murine B lymphoma system allows one to obtain a set of cell lines generated independently in vivo, andit carries significant implicationsfor the association of Ly-1 B with B lymphomas.The CHlymphomas arose from spleens b, either sponin a particular doublecongenicmousestrain, B10.H-2"-H-4 taneously or after transfer of spleen (SRBChyperimmunized) into syngeneic or F1 recipients (16). All (of 27) B lymphomas express IgM surprisingly all expressLy-1at various levels (18, 19). Moreover,although they wereelicited independently,they share idiotopes with each other and forman interconnectedfamilyof related specificities. Sevenanti-idiotype antisera to individual lymphomas weresufficient to define a series of 12 shared idiotopes expressedby 21 of the 27 CHtumors(18, 55). Antibodyspecificities of the CHlymphomas showthat they express a series of specificities reactive with phospholipidsor related determinants present on either SRBC,Bromelain-treated mouse red blood cells (BrMRBC) or E. coli (18). Furthermore, correspondingB cells present in normalspleen havebeen definedby using one anti-idiotype (present on CH12).It wasreported that cells that secrete antibodyreacting with both SRBCand BrMRBC are Ly-1 ÷, and that 50%of these cells were killed with this anti-idiotypeantisera (56). Theseresults suggesta strongrelationship between the CHlymphomas and normal Ly-1 B cells. B LYMPHOMAS FROM NFSMICEInbred Swiss strain NFSmice lack the genetic information for ecotropic murine leukemia virus (MuLV)and showa low incidence ofnonthymictumors. NFSmice congenic for MuL¥ (NFS/Nv-congenic) showmore than three-fold increase of nonthymic

Annual Reviews

206

HAYAKAWA & HARDY

lymphomas (predominantly B lineage) and myelogenous leukemias by months of age (57). Subsequently, Davidson and Morse and their colleagues found that almost all B lymphomasoccurring in NFS/Nv-congenie mice show Ly-1 expression (20). One of the more differentiated (surface Ig +) cell lines,(NFS-1) adapted to in vitro culture clearly shows antiBrMRBC specificity (17), although the specificities of other Ly-1 + B fineages cells have not been determined.

Annu. Rev. Immunol. 1988.6:197-218. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

B CELL LINES FROM SPLEEN CULTURE Ly-1 B also

show unique characteristics in culture: Whereasmost B cells from peripheral lymphoidorgans die after a short time in culture, Ly-1 B show prolonged survival. Braun found that the proliferating IgM+ B cells surviving after the crisis phase of culture all expressed low levels of Ly-1 (and, unexpectedly, lambdalight chain) (58). The generationof such lines does not exhibit strain preference; cells grow rapidly in culture and showhyperexpression and genomicamplification of c-myc(59). Morseet al also established B cell lines from spleens of BALB/cand NZBmice, and these too express Ly- 1 (60). B CELL LINES TRANSFORMEDBY BALB- AND HARVEY-MURINE SARCOMAFROM BONEMARROW OR FETAL LIVER Characteristic oncogeneexpression in Ly-1

B cells and related cell lines has not been clearly established. Most B-lineage cell lines generated by transformation with Abelson murine leukemia virus or on bone marrowstromal layers were not significantly enriched for Ly-1 expressing cells. However,it is interesting that Holmes et al found a majority of the lines transformed by various retroviruses (containing fes, ras, src oncogenes) were expressed Ly-1 (6l). These cell lines have pro- or pre-B cell phenotype, as determined by Ig-gene rearrangements and surface phenotype. Someof them had pre-B/myeloid characteristics and actually differentiated into macrophages(62, S. P. Klinken, M. A. Principato, U. R. Rapp, K. L. Holmes, J. H. Pierce, H. C. Morse, III, submitted). Such findings have important implications, suggesting a commonBand myeloid cell differentiation pathway, although it is not knownwhether this observation is limited to Ly-1 B cells. The high frequency of Ly-1 + cells mayreflect either a preferential retroviral transformation (63) in the Ly-1 + B lineage or else a relatively high frequency of Ly-1 B generation in that B-committedcell pool. However,this determination awaits further studies since the possibility of secondaryeffects due to such viruses (which might include altering the level of Ly-1 expression), can not be excluded. Certainly, their findings will provide a key to understanding Ly-1 expression on B cells and Ly-1 B.

Annual Reviews THE LYol B LINEAGE

207

PHENOTYPIC CHARACTERISTICS A major reason for concluding that Ly-1 expressing B cells form a distinct population are phenotypic observations which show that Ly-1 expression is independent of any knownantigen expression related to activation or differentiation. Furthermore, recent work suggests that Ly-1 B mayhave unique gene regulation (in addition to Ly-1 expression) not encountered in other B cells. Annu. Rev. Immunol. 1988.6:197-218. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Morphology and Unique Low CD5 Expression Ly-1 B sorted from NZBspleen or BALB/cPerC show lymphoid morphology (large nucleus and scanty cytoplasm) (33) and express most antigens commonto B cells (33). However, compared with small (high IgDexpressing) B cells, they are relatively larger, showmore internal granularity (measuredby large angle light scatter on the cell sorter) (34), are slightly more adherent to glass (34). Theyare also smaller than mitogen activated B cells and less granular or adherent than myeloid cells. Ly-1 B cells express somewhathigher average levels of surface IgMbut have much lower levels of IgD than do typical (Ly-1 -) B cells (33, 34). (IgD level not differ betweenhumanLeu-1 B cells and Leu-1 - B cells.) A determinant on B220 (detected by RA3-6B2)is also distinctively low (38). CD5(Leu-1, Ly-1) is the only one of a group of T cell differentiation antigens (in both mouse and human) found on a subset of B cells and at a uniquely low level: 10 times lower comparedto the average expression found on T cells (26, 28, 33, 39) (Figure 1). The expression of Ly-1 on the cell surface is relatively resistant to trypsin treatment (15, 64) or to modulation antibody (33).

Activation, Cell Cycle In both mouseand human, CD5-expressingB cells are not restricted to a particular stage of the cell cycle. CD5expression does not result from activation since incubation with mitogen does not induce CD5on CDSB cells (31, 32). AlthoughLy-1 B are capable of self-renewal, most cells the Ly-1 B population are not actively cycling (even amongthe reconstituting Ly-1B in transferred recipients; 37).

Differentiation:

ImmunoglobulinIsotype Expression

Almost all Ly-1 + B-lineage cells are found to express surface immunoglobulin, and few (if any) cells can be defined in situ as Ly-1 ÷ pre-B cells (17). However, we assume Ly-1 expression can occur before surface /.t expression, based on analysis of cell lines. This was demonstrated by examining clonal descendents of the NFS-5line (#-/Ly-1 - to #-/Ly-1 ÷,

Annual Reviews

Annu. Rev. Immunol. 1988.6:197-218. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

208

HAYAKAWA & HARDY

then #+/Ly-1+) (17, 32, 65) and also other Ly-1+ transformedB lineage lines (61). MostLy-1B cells expressIgM(togetherwith lowto undetectable levels of IgD), although someIgG expression was found on PerCLy-l in association with IgM(K. Hayakawa,R. R. Hardy, unpublished). While Ly-1 B are able to secrete IgGin irradiated recipients (38), such cells expressing IgG alone were belowdetection limits (<0.1%). There is clearcut evidenceat present whetherLy-1B terminally switchto expression of IgG(after deleting #), or whetherinstead transcriptional isotype regulation (66, 67) dominatesfor expression of other isotypes in Ly-1 Furthermore,the possibility remains that Ly-1 expression maybe lost uponterminal differentiation, although antibody secretion clearly can occur fromcells retaining Ly-1expression. Findings of increased 2 light chain in the normalLy-1B populationin PerC(34) also suggestuniquegeneregulation in the differentiation of Ly-1 B comparedto other B cells. This might relate to the observation that cell lines spontaneouslyarising fromspleen all express Ly-1and ;~ light chain (58) or that 2-beating immunoglobulins increase in the sera of me mice(43, 68) (wheremost B cells are Lyol+). Eventhoughthe majority Ly-1B bear x light chain, the study of light-chain expressionas it relates to B cell differentiation andB cell lineage(s) is a subject requiringfurther attention. Furthermore, several observations contradicting previous understanding about B cells havebeenmadeusing Ly-l-expressingB cell lines. /~ expression without light-chain (32, 65), non-excludedlight-chain expression (65), and VHgene replacement(69) have beendemonstrated the differentiation of Ly-1 ÷ NFS-5lines. Allelic exclusionis apparently violated on Ly-1 ÷ B cells in #-heavychain transgenic mice (69a). Such differences in immunoglobulin gene expression betweenLy-1 B and conventional B cells presumablyreflect someof the (potentially) numerous differences in the differentiation programof these cells whichwouldalso underlie the self-renewal ability of Ly-1 B. However,wemust be aware that at present, it is not clear whethersuch modesof differentiation are exclusivelylimited to cells of the Ly-1B lineage. LY-1

B (LEU-1

B)

AND AUTOANTIBODY

Anothermajor area of investigation with Ly-1 B ceils involves the study of antibody diversity. Since Ly-1B cells were initially detected in the autoimmunemousestrain, NZB,the significance of Ly-1 B was first considered to be its association with autoimmune disease. In approaching this question, studies raised further fundamentalquestions involving the relationship of B cell differentiation with antibodydiversity, since Ly-1B

Annual Reviews THE LY-1

B LINEAGE

209

Annu. Rev. Immunol. 1988.6:197-218. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

normally appear early in development, and Ly-1 B found in the adult apparently showspecificities biased towardautoantigens. Thefollowing sections summarize available evidencesuggestingrestricted specificity in Ly-1B anddiscuss the possible significanceof suchrestriction. Defective Mice: NZB and Motheaten NZBspleen cells secrete high levels of IgMincluding autoantibody(antissDNA,anti-T cell) whenincubatedin vitro. This "spontaneous"secretion (70) is often solely fromLy-1B (33) (althoughnot absolutely) (31), the autoantibodyratio (to total secreted IgM)is alwaysenrichedin Ly-1 B-derivedIgM(31). Since the total serumIgMlevel and splenic Ly-1 level roughly correlate in youngNZBadults, the Ly-1 B population in NZBmicemaycontain a higher frequencyof Ig-secreting cells than other B cells and maybe largely responsible for autoantibody secretion. However,since NZBIgMsecretion appears affected by two genetic factors, one affecting the numberof IgMsecreting cells and another the amount of secretion per cell (51), then the presenceof Ly-1B in increasednumbers mustbe consideredseparately fromtheir etiological significancein autoantibodysecretion. In fact, old NZB miceeventuallyreveal large deviationsin Ly- 1 B level, someshowingpauciclonalproliferation withoutIg-secretion. Nevertheless, although the study of the immunesystem in NZBmice involves several genetic traits uniqueto this strain, examinationof Ly-1 B in NZBmice clearly demonstratestwo distinctive capabilities of this population, namelyproliferation and high autoantibodysecretion. Motheatenmice also serve to implicate Ly-1 B with autoantibody. Abnormallyhigh levels of serumautoantibodies found in rnev (68) mice mustbe closely related to Ly-1B since almostall B cells in mev miceshow Ly-1expression(43). A series of studies by Sidmanet al showedthat such high immunoglobulin secretion seemsto be promotedby B cell-derived B cell maturationfactors uniquelyelevated in memice(71). Growthand/or differentiation factor secretion fromhumanLeu-1+ B cell lines (72) and by murineLy-1+ B cell hybridomas(73) has also beendemonstrated.It tempting to invokesecretion of such factors as a unique Ly-1 B characteristic; however,wemustadmitthat it remainsunclear whethersecretion of such factor(s) is directly responsiblefor the unusualpropertiesof CD5+ B cells (as distinguishedfromother B cells). Normal Mice: Anti-BrMRBC Specificity of + Ly-1 B Cells and of Ly-1 B Lymphomas A close relationship of Ly-1 B with autoantibodysecretion has also been demonstratedin non-autoimmune mice. Specifically, a family of auto-

Annual Reviews

Annu. Rev. Immunol. 1988.6:197-218. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

210

HAYAKAWA & HARDY

antibodies that react with bromelain-treated mousered blood cells (BrMRBC) has been found to be most closely related to Ly-1 B (31). 1966, Bussardreported detection of antibody (anti-SRBC)secreting cells in the peritoneal cavity of unprimedmice (74). Bussardand others subsequently established that most such antibody is actually anti-BrMRBC and is curiously enrichedin the peritoneal cavity as comparedwith other lymphoid tissues (75-77). Anti-BrMRBC cells can also normally detected in spleen at some frequency (78), but deliberate antigen (BrMRBC) stimulation does not increase this frequency;thus, there is memory response (78). AlthoughLPSstimulation is effective in increasing anti-BrMRBC secretion (together with other IgMautoantibodies) (7880), stimulatoryeffects dueto intestinal flora are unlikelysince germ-free mice also possess "spontaneous"anti-BrMRBC secreting cells (78). Early after LPSstimulation in vivo, sorting of splenic Ly-1 B from BALB/cmice showeda distinctive enrichment of anti-BrMRBC secreting cells fromthis minorB cell population(31). In fact, the data agree well with previous findings of anti-BrMRBC in PerC, becauseLy-1 B are also curiously enriched in PerC. Strain differences also demonstratethat the Ly-1 B frequencyparallels the level of anti-BrMRBC (34). Notably, NZB is recognizedfor high levels of anti-BrMRBC in serum(81). Introducing the xid gene into NZBmice dramatically decreases both anti-BrMRBC secretingcells (82) andLy-1B (34). Further evidenceto supportthe concept that the B cells responsible for anti-BrMRBC secretion are restricted to a subpopulation was obtained by Poncet et al (83). They found that whereasanti-BrMRBC secreting cells constitute a high frequencyof PerC B cells, the secreted antibodyconsists of a restricted familyof molecules, as determined by idiotype and V-region N-terminal sequence data of monoclonalanti-BrMRBC antibodies madefrom several different strains. The association of anti-BrMRBC specificity with Ly-1 B extends to mouseLy-1+ B cell lymphomas.It is curious that most CHseries B lymphomas(Ly-I÷) occurring in vivo show erythrocyte specificities (BrMRBC, SRBC)and/or E. coli reactivity. (Mercolino et al found in subsequentanalysis that such binding wasdue to the phosphatidylcholine determinant commonto the cell membrane;84.) This binding spectrum was also found with normalPerC B cells and provides strong supporting evidence that the origin of such B lymphomasmay not be randomly distributed amongall B cells but rather maybe restricted to Ly-1B. Normal Mice: Other Autoantibody Specificities However,the question of whether Ly-1 B is responsible for a variety of other autoantibodies in normal(BALB/c)mice has not yet been clearly resolved. This is becauseof the requirementof mitogento assay induced

Annual Reviews THE LY-1

B LINEAGE

211

Annu. Rev. Immunol. 1988.6:197-218. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

antibodysecretion (in normalmice). Other (Ly-1-) B cells dearly include cells that secrete autoantibodieswhenstimulated with a mitogen(84a; K. Hayakawa,R. R. Hardy, manuscriptin preparation). Nevertheless, wedo not consider that this observationeliminates a preferential (or probably essential) role for Ly-1B in typical physiologicalsituations wherestrong mitogenicforces are absent. Ly-1B maycontribute significantly to determiningthe level of "natural serumimmunoglobulins"(37, 38) or, in regulatoryrole in cellular interactions, to establishing the immune network (85, 86). Human Leu-1 B Theenriched frequencyof autoantibodyin CD5+ B cells wasalso evident in a study of human PBL. Although Leu-1 B in human PBLdo not spontaneouslysecrete significant levels of immunoglobulin in vitro (28), stimulation with Staphylococcusaureus induces IgMsecretion. Comparing such IgMfromcell fractions whereLeu-1B is present or absent (purifiedby FACS)shows that one autoantibody (rheumatoid factor, RF; IgM anti-IgG) is enriched 4-20-fold in the Leu-1B-containingfraction when comparedwith that in IgMderived from Leu-1- B cells (28). This high frequencyof RF specificities in Leu-1B was also demonstratedusing B cell lines transformed with EBvirus (from Leu-1+ or Leu-1- B cells) (87). The association of RFsecretion with Leu-1 B wasforeshadowed analyses showingthat increased levels of Leu-1B are found particularly amongpatients with RAor Sjogren syndrome,diseases whereRF secretion is often observed(28, 53). Althoughlevels of serumRFand Leu-1B not correlate (as for autoantibodywith Ly-1 B in older NZBmice), if secretioncomespreferentially fromsuchcells, then individualswith genetic backgroundsleading to B cell activation maybe especially prone to autoimmune disease if they also have(genetically regulated) elevated levels Leu-1 B. Ability

to Respond to Laboratory Antigens

Becauseof the rarity of Ly-1B in normaladult spleen, it is difficult to ascertain their ability to respondto typical "laboratory"antigens. At the least, it is known that the contributionof Ly-1B to secretingcells responding to mostlaboratoryantigens is low(if any) (31), in sharpcontrast their anti-BrMRBC preference. Also, the increased Ly-1 B population in NZBprovided an opportunity to purify splenic Ly-1 B (by FACS) sufficiently to eliminate other contaminating B cells and thus to test this questiondirectly. Studieson the B cells in memicealso provideinformation on Ly-1 B function since mostare Ly-1 B. Bothcases demonstrateimpaired response to such "foreign" antigens (31, 68, 88). Admittedly, these

Annual Reviews

Annu. Rev. Immunol. 1988.6:197-218. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

212

HAYAKAWA & HARDY

response deficits maybe due to preselected pauciclonality or to an altered differentiation stage in Ly-1 B that does not allow priming (as compared with other "virgin" B cells). However, the observations of Ly-1 B from both strains--sharing high autoantibody secretion and a deficit in response to foreign antigens--suggest that Ly-1 B found in adult mice very likely expresses a restricted repertoire, enriched for autoantibody spccificitics. The specificitics found in PerC Ly-1 B of normal mice already show a preference for crythrocytc related antigens. Responsesof these cells have also been tested directly using immunoglobulin allotypcs to mark cell populations in experiments where PcrC Ly-1 B reconstitute Ly-1 B in irradiated or newborn mice. Again, restricted responses wcrc found. Responses to DNPor NP coupled to TI or TD carriers wcrc impaired (Dr. P. A. Lalor, personal communication;37). It is interesting that such reconstituted Ly-1 B do respond to ~1,3 dextran (37) and that the T15 idiotype + anti-PC response apparently was enriched in Ly-1 B (Dr. A. M. Stall, personal communication). Such results suggest a functional significance for Ly-1 B immunedefense, since protective anti-PC antibody is primarily T15Id÷ (89) and dextran is a commonbacterial cell wall component.

Early B Cell Developmentand Autoanti#en Specificity Perturbation of B cells generated early in developmentcan result in disturbance of the immunerepertoire, including that of T cells (90-92). Kearneyet al demonstrated, idiotype-directed interactions are established during early ontogeny and mayhave significant influence on the adult B cell repertoire (93). Important evidence is accumulating that autoantigen reactive B cells are also generated early and appear enriched in neonatal mice (94, 95). Besides detecting such cells by direct antigen binding, family of antibody-variable regions commonto both normal fetal- and autoimmune adult B cells was indicated by measurement of idiotypebearing immunoglobulin secretion (using idiotypes directed to anti-DNA autoantibody in adult mice) (96). Thesefindings provide a theoretical basis for considering the significance of Ly-1 B and their apparent restriction to autoantibody. The Ly-1 B generated early in development probably have a higher likelihood of expressing autospecificities (or related antibodies), and such biases "early" B cells maybe reflected in the adult Ly-1 B repertoire if the Ly-1 B population maintains itself. Actually, study of humanLeu-1 B shows enrichment of RF secretion in the Leu-1 B-containing fraction from early B cells (cord blood) (28). The ability to secrete RF from Leu-1 B can seen as early as the twentieth weekof gestation.

Annual Reviews

Annu. Rev. Immunol. 1988.6:197-218. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

THE LY ol

B LINEAGE

213

Furthermore, the observation of nonrandomusage of VHgenes during early development(97, 98) has led to consideration of possible biased usage of V~gene early in B cell development;this mayrelate to enriched autoantigenspecificities (99, 100). However, wecould assumeas an alternative that the Ly-1B found in adult are a selected populationsurviving from the perinatal stage, modifiedby environmentalstimulation (which maypreferentially occur in the neonate), and mostlyconsisting of autoantigens. In such a case, the antibodydiversity present initially in Ly-1B would not be unique. Although we have not as yet reached a clear conclusion, study of Ly-1 B is deemedto be of special importance in understandingthe mechanism(s)that allow autoantibodyexpression. CONCLUDING

REMARKS

In this review wehave introduced one B cell subset found in both mouse and human,whichmaybe differentiated from the majority of B cells with regard to its progenitorand lifespan. Althoughawaitingfurther study, the characteristics associated with this B cell phenotypeare remarkableenough to suggest it maybe a uniqueB cell lineage. Theself-maintaining mechanismof Ly-1 B, experimentallydemonstratedin mice, mayprovide a clue to understandchronic B cell malignancy;it poses the vexing conceptual question of whether B CLLoriginates from a distinct B cell lineage. Furthermore,in consideration of such mechanisms,there are reports on the augmenting role of CD5on T cells (101,102),both in factor production (103, 104) and receptor expression, that permits continuousgrowth(104). This mayhelp account for the physiological distinctions of Ly-1 B (especially in contrast with "conventional"B cells). In fact, sequence analysis of CD5showinga large intracytoplasmic domainimplies that it could serve as a physiologicalgrowthregulating receptor. Onthe other hand, it is plausible to expect that B cells will be more complexand that CD5expressionmaynot be foundon all cells related to this population(38). All B cells foundearly in development as suchmust an importantsubject for further study, requiting our scrupulousattention. Thus, althoughthe functional importanceof Ly- 1 B in the immune system remainsto be clarified, its study raises fundamentalquestions concerning B cell differentiation, antibodydiversity, and tumorigenesis.Finally, we wouldventure to predict that these cells are likely to play an important role in the immune systembecauseof their presencein such phylogenetically diverse species as mouseand man. ACKNOWLEDGMENTS

Dr. Leonore A. Herzenbergand Prof. LeonardA. Herzenbergcontributed

Annual Reviews 214

HAYAKAWA& HARDY

greatly to much of the work reviewed here. Most of the FACS analyses and sorting described herein would not have been possible without the support of the Stanford FACS engineering group. Dr. M. Shimizu and Prof. T. Kishimoto made our studies with human Leu-1 B possible. We thank Dr. H. C. Morse III for sharing unpublished data with us.

Annu. Rev. Immunol. 1988.6:197-218. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Literature Cited 1. Strober, S. 1975.Immune function, cell surface characteristics andmaturation of B cell subpopulations.Transplant. Reo. 24:84-112 2. Scher, I. 1982. CBA/N immunedefective mice;evidencefor the failure of a B cell subpopulationto be expressed. Immunol.Rev. 64:117-36 3. Wortis, H. H., Burkly, L., Hughes,D., Roschelle, S., Waneck,G. 1982. Lack of matureB cells in nude micewith Xlinked immuno deficiency. J. Exp. Med. 155:903-13 4. Mond,J. J., Scher, I., Cossman,J., Kessler, S., Mongini,P. K. A., Hansen, C., Finkelman,F. D., Paul, W.E. 1982. Role of the thymusin directing the development of a subset of B lymphocytes. J. Exp. Med. 155:924-36 5. MacLennan, I. C. M., Gray, D., Kumuraratne,D. S., Bazin, H. 1982. The lymphocytesof splenic marginal zones: a distinct B cell lineage. Immunol. Today 3:305 6. Gray, D., MacLennan, I. C. M., Bazin, ÷ H., Khan, M. 1984. Migrant IgM + IgD and static IgM÷ IgD- B lymphocytessubsets. Eur. J. ltnrnunoL12: 564-69 7. Boumsell,L., Bernard,A., Lepage,V., Degos, L., Lemerle, J., Dausset, J. 1978. Somechronic lymphocyticleukemia cells bearing surface immunoglobulins share determinantswith T cells. Eur. J. ImmunoL 8:900-904 8. Boumsell, L., Coppin, H., Pham,D., Raynal, B., Lemerle,J., Dausset, J., Bernard, A. 1980. Anantigen shared by a humanT cell subset and B cell chronic lymphocyticleukemic cells. Distribution on normaland malignant lymphoidcells. J. Exp. Med.152: 22934 9. Wang,C. Y., Good,R. A., Ammirati, P., Dymbort,G., Evans, R. L. 1980. Identification of a p69, 71 complex expressed on humanT cells sharing determinantswith B-type chronic lymphatic leukemiccells. J. Exp.Med.151: 1539-44

10. Royston,I., Majda,J. A., Baird, S. M., Meserve,B. L., GrilTaths, J. C. 1980. Human T-cell antigens defined by monoclonal antibodies: the 65,000-dalton antigen of T cells (T65) is also found on chronic lymphocyticleukemiacells bearing surface immunoglobulin.J. Immunol. 125:725-31 11. Kamoun,M., Kadin, M. F., Martin, P. J., Nettleton,J., Hansen,J. A.1981.A novel human T cell antigen preferentially expressedon matureT cells andalso on (B-type) chronic lymphatic leukemiccells. J. Immunol.127:987-91 12. Martin,P. J., Hansen,J. A., Siadak,A. W., Nowinski,R. C. 1981. Monoclonal antibodies recognizing normal human T lymphocytes and malignant human B lymphocytes: a compara,tivestudy. J. Immunol. 127:1920-23 Lanier, L. L., Lynes,M.A., Haughton, 13. G., Wettstein, P. 1978. Noveltype of murine B-cell lymphoma.Nature 271: 554-55 14. Slavin, S., Strober, S. 1978. Spontaneous murineB-cell leukemia.Nature 272:624-26 15. Lanier,L. L., Warner,N. L., Ledbetter, J. A., Herzenberg,L. A. 1981. Expression of Lyt-1antigen on certain murine B cell lymphomas.J. Exp. Med.153: 998-1003 16. Lanier, L. L., Arnold, L. W., Raybourne, R. B., Russel, S., Lynes, M. A., Warner, N. L., Haughton, G. 1982. TransplantableB cell lymphomas in B10. H-2a H-4b p/rWts mice. Immuno#enetics 16:367-71 17. Hardy, R. R., Hayakawa,K., Herzenberg,L. A., Morse,H.C. III, Davidson, W.F., Herzenberg,L. A. 1984. Ly1 as a differentiation antigenon normal and neoplastic B cell. Curr. Top. Microbiol. Immunol.113:231-36 18. Pennell, C. A., Arnold,L. W., Lutz, P. M., LoCascio, N. J., Willoughby,P. B., Haughton,G. 1985. Cross-reactive idiotypes and common antigen binding specificities expressedby a series of murine B-cell lymphomas;etiological

Annual Reviews

Annu. Rev. Immunol. 1988.6:197-218. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

THE LY-I

B LINEAGE 215

implications. Proc. Natl. Acad. Sci. 29. Hokland,P., Rosenthal,P., Griffin, J. USA 82:3799-3803 D., Nadler,L. M., Dadley,J., Hokland, 19. Haughton,G., Arnold, L. W., Bishop, M., Schlossman,S. F., Ritz, J. 1983. G. A., Mercolino,T. J. 1986. TheCH Purification and characterization of series of murineBcell lymphomas: neofetal hematopoieticcells that express ÷ plastic analogues of Ly-I normal B the common acute lymphoblastic leucells. Immunol.Rev. 93:35-51 kemiaantigen (CALLA).J. Exp. Med. 20. Davidson,W.F., Frederickson,T. N., 157:114-29 Rudikoff, E. K., Coffman, R. L., 30. Ritz, J., Nadler, L. M., Bhan,A. K., Notis-McConarty,J., Pesando, J. M., Hartley, J. W.,Morse,H. C. III. 1984. A unique series of lymphomas related Schlossman,S. F. 1981. Expressionof + to the Ly-1 lineage of B lymphocyte common acute lymphoblastic leukemia differentiation. J. Immunol.133: 744antigen (CALLA) by lymphomasof cell andT-cell lineage. Blood58: 64853 21. Ledbetter,J. A., Evans,R. L., Lipinski, 52 M., Cunningham-Rundles,C., Good, 31. Hayakawa,K., Hardy, R. R., Honda, R. A., Herzenberg,L. A. 1981. EvolM., Herzenberg,L. A., Steinberg, A. utionary conservationof surface molD., Herzenberg, L. A. 1984, Ly-1 B ecules that distinguish T lymphocyte cells: Functionally distinct tymphohelper/inducer and cytotoxic/supprescytes that secrete IgMautoantibodies. sor subpopulationsin mouseand man. Proc. Natl. Acad. Sci. USA81: 2494J. Exp. Med.153:310-23 98 22. Jones, N. H., Clabby,M.L., Dialynas, 32. Hardy, R. R., Hayakawa, K. 1986. D. P., Huang,H.-J. S., Herzenberg,L. Developmentand physiology of Ly-1 A., Strominger,J. L. 1986.Isolation of B and its humanhomolog, Leu-I B. complementaryDNAclones encoding ImmunoLRev. 93:53-79 the humanlymphocyte glycoprotein 33. Hayakawa,K., Hardy, R. R., Parks, T1/Leu-1. Nature 323:346-49 D. R., Herzenberg, L. A. 1983. The 23. Huang,H.-J. S., Jones, N. H., Strom"Ly-I B" cell subpopulationin normal, inger, J. L., Herzenberg,L. A. 1987. immunodefective, and autoimmune Molecular cloning of Ly-l, a memmice. J. Exp. Med.157:202-218 brane glycoprotein of mouseT lym34. Hayakawa, K., Hardy, R. R., Herphocytesanda subset of B cells. Proc. zenberg, L. A. 1986. Peritoneal Ly-1 NatL Acd. Sci. USA84:204-8 B ceils: genetic control, autoantibody 24. Caligaris-Cappio, F., Gobbi, M., production, increased lambda light Bofill, M., Janossy,G. 1982.Infrequent chain expression. Eur. J. ImmunoL 16: 450q55 B lymphocytesexpress features of Bchronic lymphocyticleukemia.J. Exp. 35. Hayakawa, K., Hardy, R. R., HerMed. 155:623-28 zenberg,L. A., Herzenberg,L. A. 1985. 25. Le~lbetter, J. A., Rouse, R. U., Progenitorsfor Ly-1Bcells are distinct Micklem,S. H., Herzenberg, L. A. fromprogenitorsfor other B cells. J. 1980. T cell subsets defined by Exp. Med. 161:1554-68 expressionof Lyt-l,2,3 and Thy-1anti36. Hayakawa, K., Hardy,R. R., Stall, A. gens. Two-parameter immunofluorM., Herzenberg,L. A., Herzenberg,L. escence and cytotoxic analysis with A. 1986. Immunoglobulin-bearing B monoclonalantibodies modifies curcells reconstitute and maintain the rent views, d. Exp. Med.152:280-95 murine Ly-1 B cell lineage. Eur. J. 26. Bofill, M., Janossy, G., Janossa, M., Immunol. 16:1313-16 Burford,G. D., Seymour,G. J. Wernet, 37. Forster, I., Rajewsky,K. 1987. ExpanP., Kelemen,E. 1985. HumanB cell sion andfunctionalactivity of Ly-I ÷ B development. II. Subpopulationsin the cells upontransfer of peritoneal cells humanfetus. J. Imrnunol.134:1531-38 into allotype-congenic, newbornmice. 27. Antin, J. H., Emerson,S. G., Martin, Eur. J. Immunol.17:521-28 P., Gadol,N., Ault, K. A. 1986. Leu38. Herzenberg,L. A., Stall, A. M., Lalor, ÷ ÷) 1 (CD5 B cells. A major lymphoid P. L., Sidman, C., Moore, W. A., subpopulationin humanfetal spleen: Parks, D. R., Herzenberg,L. A. 1986. phenotypicand functional studies. J. TheLy-1B cell lineage, lmmunol.Rev. Immunol. 136:505-10 93:81-102 28. Hardy,R. R., Hayakawa,K., Shimizu, 39. Hayakawa, K., Hardy, R. R., HerM., Yamasaki,K,, Kishimoto,T. 1987. zenberg,L. A., Steinberg, A. D., HerRheumatoid factor secretion from zenberg, L. A. 1984. Ly-1 B: A func+ humanLeu-I Bcells. Science236: 81tionally distinct B-cell subpopulation. 83 In Progress in ImmunologyV., eds.,

Annual Reviews

Annu. Rev. Immunol. 1988.6:197-218. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

216

HAYAKAWA& HARDY

Y. Yamamura,T. Tada, pp. 661-68. Tokyo: Academic 40. Hayakawa,K., Ishii, R., Kishimoto, T., Herzenberg, L. A., Hardy, R. R. 1987.Localizationof Ly-1Bcells in the peritoneal cavity: Genetic control of frequency. In NewHorizonsin Animal Modelsfor AutoimmuneDisease, ed. M. Kyogoku,H. Wigzell. Tokyo:AcademicPress. In press 41. Green, M. C., Shultz, L. D. 1975. Motheaten, and immunodeficient mutantof the mouse.I. Genetics and pathology. J. Hered.66:250-58 42. Shultz, L. D., Coman,D. R., Bailey, C. L., Beamer,W.G., Sidman,C. L. 1984. "Viablemotheaten,"a newallele at the motheatenlocus. I. Pathology.Am.J. Pathol. 116:179-92 43. Sidman,C. L., Shultz, L. D., Hardy, R. R., Hayakawa,K., Herzenberg, L. A. 1986. Production of immunoglobulin isotypes by Ly-1÷ B cells in viable Motheaten and normal mice. Science 232:1423-25 44. Amsbaugh,D. F., Hansen, C. T., Prescott, B., Stashark,P. W.,Barthold,D. R., Baker, P. J. 1972. Geneticcontrol of the antibody response to type III pneumoeoeealpolysaeeharide in mice. J. Exp. Med.136:931-49 45. Scher, I., Sharrow,S. O., Paul, W.E. 1976. X-linkedB-lymphocyte defect in CBA/Nmice. III. Abnormal developmentof B-lymphocytepopulations defined by their density of surface immunoglobulin. J. Exp. Med. 144: 507-18 46. Hardy, R. R., Hayakawa,K., Parks, D. R., Herzenberg, L. A. 1983. Demonstration of B cell maturationin Xlinked immunodeficientmice by simultaneous three colour immunofluorescence. Nature 306:270-72 47. Hardy, R. R., Hayakawa,K., Parks, D. R., Herzenberg,L. A., Herzenberg, L. A. 1984. Murine B cell differentiation lineages. J. Exp. Med.159: 1169~88 48. Marcos, M. A. R., De La Hera, A., Gaspar,M.L., Marquez,C., Bellas, C., Mampaso,F., Toribio, M. L., Martinez, A. C. 1986. Modificationof emerging repertoires by immunosuppression in immunodeficient mice results in autoimmunity.Immunol.Rev. 94: 5174 49. Manohar,V., Brown,E., Leiserson, W. M., Chused,T. M. 1982. Expressionof Lyt-1 by a subset ofB lymphoeytes.J. Immunol.129:532-38 50. Raveche,E. S., Alabaster,O., Tjio, J. H., Taurog,J., Steinberg, A. D. 1981.

Analysis of NZBhyperdiploid spleen cells. J. ImmunoL 126:154-60 51. Manny,N., Datta, S. K., Schwartz,R. S. 1979. Synthesis of IgMby cells of NZBand SWR mice and their crosses. J. lmmunol.122:1220-27 52. Hardy,R. R., Yamasaki,K., Ishii, R., Kishimoto, T., Hayakawa,K. 1987. Differences in lymphocytepopulations of murine autoimmunestrains demonstrated by multiparameter FACS analysis. In NewHorizons in Animal Models for AutoimmuneDisease, ed. M. Kyogoku,H. Wigzell. Japan: Academic.In press 53. Plater-Zyberk, C., Maini, R. N., Lam, K., Kennedy,T. D., Janossy, G. 1985. A rheumatoidarthritis B cell subset expresses a phenotypesimilar to that in chroniclymphocyticleukemia.Arth. Rheum.28:971-76 54. Fox, R. I., Chen, P., Carson, D. A., Fong, S. 1986. Expressionof a crossreactive idiotype on rheumatoidfactor in patients with Sjogren’s syndrom.J. ImmunoL136:477-83 55. Pennell, C. A., Arnold, L. W., LoCascio, N., Lutz, P. M., Willoughby, P. B., Haughton,G. 1984. The CHseries of murine B cell lymphomas:Identification of cross-reactive idiotypes and restricted antigen specificities. Curr. TopicsMicrobiol. ImmunoL113:251-57 56. Bishop, G. A., Haughton,G. 1985. H2 control of expressionof an idiotype shared by normalB cells and a B-cell lymphoma,lmmunotTenetics21:355456 57. Fredrickson, T. N., Morse,H. C. III, Rowe, W. P] 1984. Spontaneous tumors of NFS mice congenic for exotropic marineleukemiavirus induction loci. J. NatLCancerlnst. 73: 52124 58. Braun, J. 1983. Spontaneousin vitro occurrence and long-term culture of murine B lymphoblast cell lines. J. lmmunoL130:2113-20 59. Citri, Y., Braun,J., Baltimore,D.1987. Elevated myc expression and c-myc amplification in spontaneouslyoccurring Blymphoid cell lines. J. Exp.Med. 165:1188-94 60. Morse,H. C. III, Lamers,M.C., Rudikoff, E. K., Davidson, W. F. 1984. Characteristics of continuousin vitro lines of Balb/c andNZBB ceils. Curt. Top. Microbiol. Immunol.113:210-16 61. Holmes,K. L., Pierce, J. H., Davidson, W.F., Morse,H. C. III. 1986. Murine hematopoieticcells with pre-B or preB/myeloidcharacteristics are generated by in vitro transformationwith retro-

Annual Reviews

Annu. Rev. Immunol. 1988.6:197-218. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

THE LY-I viruses containingfes,ras, able andsrc oncogenes.J. Exp. Med.164; 443-57 62. Davidson, W. F. 1987. In Mechanism of B Cell Neoplasia,ed. F. Melchers, M.Potter. Basel, Switzerland:Roche 63. Pierce, J. H., Aaronson,S. A. 1982. Balb- and Harvey-mudne sarcoma virus transformationof a novellymphoid progenitor cell. J. Exp. Med.150: 873~87 64. Ledbetter, J. A., Herzenberg, L. A. 1979. Xenogeneicmonoclonal antibodies to mouse lymphoid differentiation antigens, lrnmunol.Rev. 47: 63-90 65. Hardy, R. R., Dangl, J. L., Hayakawa,K., Jaeger, G., Herzenberg,L. A., Herzenberg, L. A. 1986. Frequent lambdalight chain gene rearrangement and expression in a Ly-1 B lymphoma with a productivekappa chain allele. Proc. Natl. Acad.Sci. USA83:1438-42 66. Braun,J., Citri, Y., Baltimore,D., Forouzanpour, F., King, L., Teheranizadeh, K., Bray, M., Kliewer, S. 1986. B-Lyl cells: Immortal Ly-1+ B lymphocytecell lines spontaneously arising in murine splenic cultures. Immunol.Rev. 93:5-21 67. Chert, Y-W.,Word,C., Dev,V., Uhr, J. W., Vitetta, E. S., Tucker,P. W.1986. Doubleisotype production by a neoplastic B cell line. II. Allelically excludedproductionof # and),~ heavy chain without CHgene rearrangement. J. Exp. Med. 164:562-79 68. Shultz, L. D., Green, M. C. 1976. Motheaten, an immunodeficient mutant of the mouse. II. Depressed immune competence and elevated serum immunoglobulins.J. Immunol. 116:936-43 69. Kleinfeld, R., Hardy,R. R., Tarlington, D., Dangl,J., Herzenberg,L. A., Weigert, M. 1986. Recombination betweenan expressed immunoglobulin heavychain gene and a germlinevariable gene segmentin a Ly-1÷ B-cell lymphoma.Nature 322:843-46 69a. Herzenberg,L. A., Stall, A. M., Braun, J., Weaver,D., Baltimore, D., Herzenberg, L. A., Grossehedl, R. 1987. Development of the predominantB cell lineage is impairedin immunoglobulin heavychain transgenicmice.Nature.In press P. J., Dixon,F. 70. Izui, S., McConahey, J, 1978. Increased spontaneouspolyclonal activation of B lymphocytesin mice with spontaneous autoimmune disease. J. Immunol.121:2213-19 71. Sidman, C. L., Marshall, J. D., Masiello,N. C., Roths, J. B., Shultz,

B LINEAGE 217

L. D. 1984. NovelB-cell maturation factor from spontaneously autoimmuneviable motheatenmice. Proc. Natl. Acad. Sci. USA81:7199-7202 72. Kawamura, N., Muraguchi,A., Akira, H., Horii, Y., Mutsuura,S., Hardy,R. R., Kikutani, H., Kishimoto,T. 1986. A case of human B cell leukemia implicating an autocrine mechanism in the abnormal of Leu-1B cells. J. Clin. Inv. growth 78:1331-38 73. Sherr, D. J., Braun, J., Dorf, M. E. 1987. Ly-I B helper cell hybridomas (BH) produce anti-idiotype and nonimmunoglobulin lymphokine(s). Immunol. 138:2057-62 74. Bussard, A. E. 1966. Antibody formarionin non immune peritoneal cells after incubation in gumcontaining antigen. Science 153:887-88 75. Pages, J., Bussard, A. 1975. Precommitment of normal mouse peritoneal cells by erythrocyteantigensin relation to autoantibody production. Nature 257:316-17 A. J. 1978. 76. Steele, E. J., Cunningham, Highproportion of Ig producingcells makingautoantibody in normal mice. Nature 274:483-87 77. Lord, E. M., Dutton, R. W.1975. The properties of plaque-forming cells from autoimmune and normal strains of mice with specificity for autologous erythrocyte antigens. J. Immunol.115: 1199-1205 78. Cunningham, A. J. 1974. Large number " of cells in normalmiceproduceantibodycomponentsof isologous erythrocytes. Nature252:749-51 79. Hammarstrom,L., Smith, E., Pdmi, D., Moiler, G. 1976. Induction of autoantibodies to red blood cells by polyclonal B cell activators. Nature 263:60-61 80. Hang,L., Slack, J. H., Amundson, C., Izui, S., Theofilopolous,A. N., Dixon, F. J. 1983. Induction of murineautoimmunedisease by chronic polyclonal B cell activation. J. Exp. Med.157: 874~83 81. DeHeer,D. H., Linder, E. J., Edgington, T. 1978. Delineation of spontaneous erythrocyte autoantibody responses of NZBand other strains of mice. J. Immunol.120:825-30 82. Rosenberg, Y. J. 1979.Influenceof the sex-linked defect in CBA/N mice on autoimmuneresponses to isologous erythrocytes. J. Exp. Med.150:1561-66 83. Poncet, P., Kocher,H. P., Pages, J., Jaton, J-C., Bustard, A. E. 1985. Monoelonal autoantibodies against mouse red bloodcells: Afamilyof structurally

Annual Reviews

Annu. Rev. Immunol. 1988.6:197-218. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

218

HAYAKAWA & HARDY

restricted molecules.Mol.Immunol.22: 541-51 84. Memolino, T. J., Arnold, L. W., Haughton,G. 1986. Phosphatidylcholine is recognizedby a series of Ly-1+ murine B cell lymphomas specific for erythrocyte membranes.J. Exp. Med. 163:15~65 84a. Conger,J. D., Pike, B. L., Nossal, G. J. V. 1987Clonalanalysis of the antiDNArepertoire of murine B lymphocytes. Proc. Natl. Acad.Sci. USA. In Press 85. Okumu~:a, K., Hayakawa,K., Tada, T. 1982.Cell-to-cell interactioncontrolled by immunoglobulingenes. Roles of Thy-l-, Lyt-1÷, Ig÷ (B’) cell in allotype-restricted antibodyproduction.J. Exp. Med. 156:443-53 86. Sherr, D. H., Dorf, M.E. 1984. Idiotype specific helper population which bears immunoglobulin,Ia, and Lyt-1 determinants.J. Exp. Med.159: 11891200 87. Casali, P., Burastero,S. E., Nakamura, M., Inghirami,G., Notkins,A. L. 1987. Humanlymphocytes making rheumatoid factor and antibody to ssDNA belongto Leu-1 + B-cell subset. Science 236:77-81 88. Sidman,C. L., Shultz, L. D., Unanue, E. R. 1978. The mouse mutant "Motheaten".If. Functionalstudies of the immunesystem. J. Itnmunol. 121: 2399-2404 89. Briles, D. E., Forman,C., Hudak,S., Claflin, J. L. 1982. Antiphosphorylcholine antibodies of the T15 idiotype are optically protective against streptococcus pneumoniae. J. Exp. Med. 156:1177-85 90. Black, S. J., Herzenberg,L. A. 1979. B-cell influences on the induction of allotype suppressor T cells. J. Exp. Med. 150:174-83 91. Bottomly, K., Janeway, C. A. Jr., Mathieson,B. J., Mosier, D. E. 1980. Absenceof an antigen-specifichelper T cell required for the expressionof the T15idiotype in micetreated with anti/t antibody.Eur. J. Immunol.10:159453 92. Hayglass,K. T., Benacerraf,B., Sy, M. S. 1986. T cell developmentin B celldeficient mice.V.Stoppinganti-# treatmentresults in Igh-restricted expansion of the T suppressorcell repertoire concomitant with the developmentof norreal immunoglobulinlevels. J. Exp. Med. 164:36-49 93. Kearney,J. F., Vakil, M. 1986. Idio-

type-directedinteractions during ontogenyplay a majorrole in the establishmentof the adult B cell repertoire. lmmunol.Rev. 94:39-50 94. Dighiero, G., Lymberi,P., Holmberg, D., Lundquist,I., Coutinho,A., Avrameas,M.1985. Highfrequencyof natural autoantibodies in normalnewborn mice. J. Immunol.134:765-71 95. Vakil, M., Kearney,J. F. 1986. Functional characterization of monoelonal auto-anti-idiotype antibodies isolated from the early B cell repertoire of BALB/cmice. Eur. J. ImmunoL16: 1151-58 96. Datta, S. K., Stollar, D. B., Schwartz, R. S. 1983. Normalmice express idiotypes related to autoantibody idiotypes of lupus mice. Proc. Natl. Acad. Sei. USA80:2723-27 97. Yancopouios,G. D., Desiderio, S. V., Paskind,M., Kearney,J. F., Baltimore, D., Alt, F. W.1984.Preferential utilization of the most JH-proximal VH gene segments in pre-B cell lines. Nature 292:629-31 98. Permutter, R. M., Kearney, J. F., Chang,S. P., Hood,L. E. 1985. Developmentally controlled expression of immunoglobulin VHgenes. Science 227: 1597-1601 99. Painter, C., Monestier,M., Bonin,B., Bona, C. A. 1987. Functional and molecularstudies of V genes expressed in autoantibodies. Immunol.Rev. 94." 75-98 I00. Manheimer-Lory,A. J., Monestier, M., Bellon, B., Alt, F. W., Bona, C. A. 1987. Fine specificity, idiotypyand nature of cloned VHgenes of murine monoclonal rheumatoid factor antibodies.Proc.Natl. Acad.Sci. In press 101. Hollander,N. 1982. Effects ofanti-Lyt antibodies on T-cell functions. ImmunoL Rev. 68:43q56 102. Logdberg, L., Shevach, E. M. 1985. Roleof the Ly1 antigen in interleukin 1-inducedthymocyte activation. Eur.J. Immunol.15:1007-13 103. Stanton,T., Stevens,T. L., Ledbetter, J. A., Wofsy,D. 1986. Anti-Ly-1antibodyinducesinterleukin 2 release from T cells. J. lmmunol.136:1734-37 104. Ledbetter,J. A., Martin,P. J., Spooner, C. E., Wofsy,D., Tsu,T. T., Beatty,P. G., Gladstone,P, 1985. Antibodiesto Tp67 and Tp44 augment and sustain proliferative responsesof activated T cells. J. lmmunoL 135:2331-36

Annu. Rev. Immunol. 1988.6:197-218. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Annu. Rev. Immunol. 1988.6:197-218. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Annual Reviews www.annualreviews.org/aronline Ann.Rev.lmmunol. 1988.6." 219-49 Copyright ©1988by Annual ReviewsInc. All rightsreserved

Annu. Rev. Immunol. 1988.6:219-249. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

OPIOID PEPTIDES AND OPIOID RECEPTORSIN CELLS 1OF THE IMMUNE SYSTEM Nicholas E. S. Sibin#a* and Avram Goldstein~f Departmentsof * Medical Microbiologyand ? Pharmacology, Stanford University, California 94305 INTRODUCTION Numerous opioid peptides are encodedby three different genes(1-5). Four types of opioid receptor (#, 6, x, 5) havebeenclearly identified but have ~ Abbreviations used: ACTH, adrenocorticotropic hormone;ADCC, antibody-dependent cellular cytotoxicity; B~ax,total numberof binding sites; BREM, bremazocine,an agonist with modestselectivity for ~¢ sites; CCK,cholecystokinin;ConA,concanavalinA; DADLE, [D-Ala~,D-LeuS]enkephalin, an agonist somewhatselective for 6 ~ites; DEX,dextrorphan, inert enantiomer of LEV;DHM,dihydromorphine,a #-selective agonist; DIP, diprenorphine, a relatively nonselective antagonist; DYN, dynorphin,a peptide product of the dynorphingene, selective for x sites; EKC,ethylketazocine, a benzomorphan agonist with modest selectivity for x sites; END,endorphln; a peptide product of the pro-opiomelanocortingene;~t- and),-endorphinare fragmentsof/~-endorphin;~-endorphin is selective for ~ sites; ETO,etorphine, a relatively nonselective agonist; fMLP,formyl-methionyl-leucinyl-phenylalanine;granulo,granulocyte(s);h, human;IC50,concentrationof a competing ligand that reduces specific binding of radioligand by 50%;icy, intracerebroventricular; IFN, interferon; ir -~, immunoreactive; Kd, Ki, equilibriumdissociation constants, determinedby binding isotherm or by competition, respectively; LENK,[Leu]enkephalin, a minorproductof the pro-enkephalingene, an agonist somewhat selective for 6 sites; leuko, leukocyte(s);LEV,levorphanol,a #-selectiveagonist; lympho,lymphocyte(s); mouse;M~b,macrophage(s);MENK, [Met]enkephalin,major product of the pro-enkephalin gene, a 6-selectlve agonist; MNC, mononuclearcell(s); mono,monocyte(s);MOR, morphine, a/z-selective agonist; NA,not applicable; NAL,naloxone,a moderatelytz-selective antagonist; NK,natural killer; NR,not reported; NTX,naltrexone, a long-lasting #-selective antagonist closely related to naloxone; PBL,peripheral blood lymphocyte(s); PBMNC, peripheral blood mononuclearcell(s); PHA,phytohemagglutinin; PMNL,polymorphonuclear leukocyte(s); r, rat; RIA,radioimmunoassay;spleno, splenocyte; SRBC,sheep red bloodcells; VIP,vasoactiveintestinal peptide.

0732-0582/88/0410-0219502.00

219

Annual Reviews

Annu. Rev. Immunol. 1988.6:219-249. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

220

SIBINGA & GOLDSTEIN

not yet been cloned. Opioid peptides, opioid receptors, and evidence of opioid function havebeenreported not only for the central andperipheral nervoussystemsbut for manynon-neuraltissues as well (4, 6-9). Theearliest report of opioid effects on cells of the immune systemwas that of Wybranet al in 1979(10). At nanomolarconcentrations opioids were shownto affect active rosetting of humanT lymphocytes. Two characteristics of opioid systems were noted in this study--stereospecificity and reversal by naloxone (NAL).Furthermore, as morphine (MOR)inhibited rosetting whereas [Met]enkephalin (MENK) stimulated it (both effects blocked by NAL),multiple opioid receptor types were implicated. This review covers the subsequentliterature, through early 1987. The exciting prospect of linking immunoregulation to neural opioid systems has emergedduring this period (11-20), and numerousstudies have expandedon the findings of Wybranet al. To assess the evidence we Table 10pioid binding

Reference 37

Species, cell type

41

h, granulo & mono m, spleno membranes h, lympho& platelets h, T-lympho line (Jurka0 h, B- & T-lympho b, PMNL

44

h, PBMNC

42 38 39 40

43

108

h,

PMNL

guinea-pig, brain membranes

Radioligand

Standard binding assay?

Saturability

DHM

yes: isotherm

yes

MENK

NR

NR

NAL

yes, competition

yes

LENK

yes, isotherm& competition yes, incomplete

yes

yes, incomplete, NAL,DIP, EKC, DADLE no isotherm NAL, DADLE yes, isotherm

yes

fiEND

NR

ETO

DHM, DAME, EKC, NAL

no; radioreceptor time course, autoradiography, internalization, morphologicchanges yes

yes

no binding

NR

Annual Reviews OPIOIDS

IN THE IMMUNE SYSTEM 221

formulatedspecific criteria for evaluation, using a list of well-established characteristics shared by knownopioid systems. Weclassified each paper into one of three major categories of investigation--opioid binding, functional assays of opioid activity, and opioid peptide production--and we then analyzed the data according to criteria developed for that category.

OPIOID BINDING Annu. Rev. Immunol. 1988.6:219-249. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Thefollowing criteriawereusedto evaluatebinding studies,andthese comprise the principal columnheadings of Table 1.

Radioligand Until recently, opioid radioligands distinguished poorly amongthe several types of opioid binding sites (21-23). Recognition of the multiplicity opioid receptors (24-27) led to the developmentof radioligands relatively

Pattern of affufities of competing ligands

Stereospecificity

Further characterization

claimed (no data) NR

NR

3000-4000sites per cell K~ approx 10 nM

NR

K~ 0.59 nM(no data)

NR

MOR only

NR

NR

NR

partial

NAL, MORdo not compete to 15-30 mM complex, DADLE, EKC fiEND, etc. at/~M cone. NR

NR

NR

NR

DIP = fiEND in blocking binding and internalization

no specific binding, even with PHAor ConAstimulation; sensitivity about 2000sites per cell at 0.1 nMradioligand, 100 molecules bound per cell; binding variable between individuals

NR

NR

NR

NR 1200 NALsites/cell, 5 times more EKCsites, no DADLEbinding

IL-1 concentration not stated, unconventional analysis of binding inhibition by IL-1

Annual Reviews

222

SIBINGA & GOLDSTEIN

selective for one or another receptor type. Thevarious radioligands used in the studies reviewedhere are noted in Table 1 and are describedin the list of abbreviations.Noneof themis sufficiently selective to label a single type of bindingsite exclusively, and all of themhavebeenreplaced today by moreselective ones.

Annu. Rev. Immunol. 1988.6:219-249. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Standard Bindin# Assay Astandard binding assay begins with a saturation isotherm to determine that there are indeedsaturable sites, to measurethe extent of nonsaturable (nonspecific)binding, to find the high-affinity Ka, andto estimate Bmax for the high-affinity site. Competition by unlabeledligandsagainst a fixed low concentrationof radioligand yields IC~0values, fromwhichKi values are computed,and a quantitative rank order of affinities is constructed. These elementsof a standard binding assay are the sine qua non for claimingthe existenceof opioidbindingsites.

Saturability It is essential to demonstrate that a significant part of the observedbinding of a radioligandis dueto a limited numberof specific bindingsites, as by competitionor saturation analysis. In competitionstudies, increasing the concentration of an unlabeled competingligand diminishesthe binding of a constant amountof radioligand. In saturation analysis, the binding isotherm represents the difference in radioligand binding in the absence and presence of sufficient unlabeled competingligand over a series of radioligand concentrations; a plateau at the higher concentrations indicates that the bindingsites are saturable (28-31).

Stereospecificity Opioidreceptors showa striking degree of stereospecificity for (-) enantiomers, e.g. for LEVover DEX,a pair used frequently since their introductionfor this purposeby Goldsteinet al (28) in 1971. Pattern of Affinities of Competin9 Ligands Multipletypes of opioid receptors are known,with well-established patterns of affinity for various ligands. Opioidbindingto cells of the immune systemmaybe expectedto display characteristics consistent with one or another knowntype(s) of opioid receptor. Thequantitative rank order affinity of competingligands in competition assays using several typeselective radioligandscan establish whattype of opioidreceptor is in hand (32).

Annual Reviews

OPIOIDSIN THE IMMUNE SYSTEM223

Annu. Rev. Immunol. 1988.6:219-249. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Further Characterization Theeffects of structural modification on opioid binding have been well established: (a) Opioidbinding (and also pharmacologicactivity) of opioid peptides requires an intact NHE-terminal tyrosine. Acylatingor otherwiseblocking the ~-aminogroup or blockingits phenolic hydroxyl(or, by analogy, the 3-OHof a morphinan)drastically reducesaffinity (33, 34). (b) + diminishes agonist bi nding wh ile le aving antagonist (e.g. NAL) bindinglargely unaffected(35). (c) Guaninenucleotides diminishbindingof manyagonists while affecting antagonist binding to a lesser extent; whenNa+ is also present this differenceis enhanced (3 l, 36). (d) Bindingof a ligand to its preferred type of opioidreceptor is often characterized by an equilibrium dissociation constant in the low nanomolar or subnanomolar range, i.e. high-affinity binding. Opioid Bindin9 Studies Table 1 summarizes eight studies on binding to opioid receptors on cells of the immunesystem and an additional study on the effects of IL-1 on opioid binding in guinea-pig brain membranes.Of the eight, seven found evidencethat the authorsinterpreted as specific opioid binding. In five of these, somedegree of saturability was observed(37-41). The essential demonstrationof stereospecificity was undertakenin only two; one of these presentedno relevant data (37), and in the other the stereospecific difference betweenenantiomersof a single competingligand (NAL)was muchless than one ordinarily sees (41). Apanel of ligands was employed in only one study (40); the complexresults suggested multiple receptor types but did not conformto any recognizable pattern. Although two groups mentioned dissociation constants in the nanomolarrange and stated the numberof bindingsites per cell, no supportingdata wereoffered (37, 42). Onepaper (4 l) presenteddata fromwhichthe numberof occupied sites per cell could be calculated--1200with NALas radioligand, about 6000with EKC--butas no saturation isotherm was shown, the degree of site occupancyis unknown, so there is no wayto estimate Bronx,the actual total number of sites. Anautoradiographic approach (43) suggested binding and internalization of fiEND(fi endorphin)specifically labeled with 125I at a position that does not interfere with opioid receptor binding. Theligand wasinternalized, and both binding and internalization wereinhibited by unlabeled fiENDor by DIP. Bindingwas estimated at only 100 moleculesper cell, and the authors correlated this with changein cell shape. However,as

Annual Reviews

Annu. Rev. Immunol. 1988.6:219-249. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

224

SIBINGA

& GOLDSTEIN

neither a binding isotherm nor conventional competition curves were obtained, the significanceof the findings remainsuncertain. Twofeatures are noteworthyin the binding studies that yielded positive results. First, the data are tentative or fragmentary,of a kind that would normally characterize a preliminary communication,yet no follow-up reports haveappeared. This leaves roomfor speculation that the initial findings could not be fully replicated andextended. Second,in several of the papers the experimentalproceduresand methodsof data presentation and analysis are distinctly unconventional, makinginterpretation difficult. In the investigation that wejudge to have conformedmost closely to standard binding technique and modeof analysis (44), samplesfromfive subjects were used. Onthe whole, no specific opioid binding wasfound in resting or mitogen-stimulatedmononuclear cells at an apparent detection limit of a few thousandsites per cell. However,these authors did observe saturable binding in cells from someindividuals. Theydiscounted this, regardingit as spurious--but for no very compellingreason. As individual variation wouldbe of considerableinterest and mightevenexplain negative or variable results obtainedin studies with smallsamples,it is unfortunate that the authorsdid not explorethis point further. One of us (A. Goldstein, unpublished) recently tested 18 different human,mouse,and bovinecell lines, using techniques well-validated for opioid receptors on brain membranes (45). Assayshighly selective for 3, and x sites were employed.A variety of cell types wasrepresented, including cytotoxic and helper T lymphocytes,macrophages,endothelial cells, mastcells, andseveral precursormyeloidlines, All cells werecultured underoptimal growthconditions for the particular line, without mitogen or antigen stimulation. Exhaustivelywashedmembrane preparations from cell homogenateswere used, as for receptor binding studies with brain. Nospecific opioid bindingwasfound. Thedetection limit varied according to the radioligandused andthe number of cells available; the highest limit was200sites per cell. Summary Wefind the case for specific opioid binding to be unconvincing.Yet we knowfrom the pharmacologicevidence (see below) that somecells of the immunesystem do indeed have opioid receptors. It would seem worthwhile, therefore, to study such cells, under conditions in whichthey are knownto be respons.ive to opioids in a naloxone-reversiblemanner,rather than to choose ceils at randomfor binding assays. Weknowof no rules that specify howmanyreceptors are required to producea sufficient signal. Somelymphokine receptors are present at very low density (a few hundred

Annual Reviews OPIOIDS IN THE IMMUNE SYSTEM

225

Annu. Rev. Immunol. 1988.6:219-249. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

or less per cell) in cells in whichtheyare clearly functional(46-50).Should opioid receptors also be present in small numbers, and should ligand affinities be as high as in neural tissues, moresensitive assays (perhaps employing125I-labeled ligands) will be required to detect themwith certainty. Alternatively, there maybe special requirements,as yet undefined, for the expressionof opioid receptors. As notedin the final section of this review, the enkephalingene is expressedin T helper lymphocytes only after activation by lectins or antigens; possibly receptor expression requires similar activation, but no studies haveaddressedthis question. FUNCTIONAL

ASSAYS OF OPIOID

ACTIVITY

Thefollowingcriteria wereused to evaluate functional studies, and these comprisethe principal columnheadingsof Table 2. Agonists and Patterns of Agon&t Potencies Different opioid agonists havewidelydiffering potencies. Parallel assays with panelsof agonistshaveelicited distinct patterns(i.e. quantitativerank orders) of potencyin assay systemsusing different responsive tissues. Disparaterank orders of potencyof the samepanel of agonists in different tissues haveconstituted an importantargumentfor the existence of multiple types of opioid receptors (24). Thepattern can suggestwhichtype receptor is likely to be mediating an observedeffect. Other meansof differentiating receptor types in pharmacologic assays havebeenselective inactivation (32, 51) andinductionof selective tolerance (52). Dose-Response Relationship Pharmacologicallymediated actions of agonists produce characteristic sigmoidaldose-responsecurveswheneffect is plotted against the logarithm of the dose or concentration. Byconvention, in biochemistryand pharmacology, concentrations increase fromleft to right, opposite to the customaryplotting of dilutions in the immunology literature. The maximal effect of an agonistis reflected in a plateau of the dose-responsecurve. A decreasein effect at doses higher than those producinga plateau is anomalous, suggesting the presence of multiple receptors mediatingopposite effects. Themaximaleffect of an agonist is analogousto the saturation of binding sites described earlier; however,as agonist potencydependsnot only on affinity for the receptorbut also on intrinsic activity andreceptor reserve, there need be no exact equivalence betweenreceptor occupancy andbiologiceffect.

Annual Reviews Table2 Functional assays of opioid activity

References

Species, cell type

Annu. Rev. Immunol. 1988.6:219-249. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Chemotaxis 56 Ix, PBMNC & PMNL

Effect, assay

chemotaxis,effect on mono,small effect on PMNL

fiEND, MENK

BEND > DADLE = DYN > levallorphan > BREM>> MOR fiEND

57

h, PBMNC

chemotaxis

58

h, PMNL

59

h, PMNL

enhancedchemotaxis to fMLP changeof cell shape

122

h, ConAinhibit productionof stimulated mono T-lymphochemotactic factor h, leuko chemotaxis

60

Macrophage and granulocyte activity 62 r, peritoneal increased ADCC, Mq5 superoxide production, cGMP 63 r, peritoneal increased Na & Ca flux, cGMP,cAMP M~b 64 123 124

125

increased superoxide h, PMNL, peritoneal M~b production enhanced IFNv h, PBMNC production by ConAstimulatedcells h, PMNL inhibition of fMLPstimulated superoxide production m, peritoneal IFN~,,-induced Mq~ tumodcidalactivity

Effects onmastcells 65 r, pleural & reverseinhibition peritoneal mast of IgE-mediated cells serotonin release 66 r, peritoneal enhancedCa uptake mastcells whealand flare 67 h, skin test elicited 126

r, guinea-pig skin

Agonist(s)and pattern of agonist potencies

dye extravasation

fiEND > LEV

MENK

MENK

DYN > fiEND > MOR fiEND, MENK

LENK, MENK

no effect with fiEND, LENK, MENK, ~END LEV > MOR

fiEND > LEV = MOR DYN> fiEND > DADLE = morphiceptin = MOR = codeine~- meperidine DYN,substance P

Annual Reviews

Annu. Rev. Immunol. 1988.6:219-249. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Dose-response relationship

Stereospecificity

Further characterization

Antagonism

bimodal

NR

yes, NALon PBMNC

normal,but less effect at > 10 nM

yes

yes, NAL (stereospecific)

ball-shaped

NR

yes, data incomplete

yes

yes, NAL(but see tex0 yes, DIP

bimodal

NR

no, NALwas agonist

NR

NR

yes, NAL

NALantagonized g-gliadin inhibition of chem6taxis

ball-shaped

NR

yes, NAL

bell-shaped

NR

yes, NAL

yes, M~;bellshaped, PMNL unclear

NR

yes, NAL

NR

no, NAL

stimulate Fcmediatedactivities; role of calmodulin? opioid mechanism only at low concentrations DYN,fiEND at 1 pMon PMNL,#M on M~b donorvariability

unclear

NR

NR

> I00 nM LENKor MENKrequired

NA

NA

NA

NR

yes, normal

yes, yes, NAL LEV/DEX

yes, normal

yes, yes, NAL LEV/DEX NR yes, NALvs. MOR

yes, normal NR

NR

effects @pMcone.; icy fiENDcauses in vivo migration of M~b-like cells max.effect @10 pM

peak effect @1 nM effect comparable to fMLP effective @pM

opioidsblockeffect of PGEI,isoproterenol, dopamine 1-10 #Mrequired! insufficient NAL used vs. DYN

no, but only implicates amine one NALdose release tried

Annual Reviews

228

SIBINGA & GOLDSTEIN

Table2 (continued)

Annu. Rev. Immunol. 1988.6:219-249. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

References

Species, cell type

Effect, assay

Lymphocyte proliferation and function immunosuppression, 42 m, cultured spleno plaque formation with SRBC, DNP-Ficoll 68 h, B lympho immunosuppression, plaque formation with ovalbumin 40 proliferation, m, PBL h, T lympho thymidine incorporation

Agonist(s)and pattern of agonist potencies

~END = LENK = MENK at 50 nM ~END

fiEND

127

h, lympho

PHA-stimulated proliferation, thymidine incorporation

fiEND

128

h, PBL

fiEND,other peptides

129

h, PBL

123

h, PBMNC

enhancedmercuric chloride-stimulated proliferation induction of "suppressorcell" activity enhancedproduction of IFNy active rosetting with SRBC

MOR

inhibitionof total rosetting with SRBC active rosetting with SRBC active rosetfing with SRBC active rosetting with SRBC

fiEND = MOR= MENK = LENK MENK = LENK

T-cell rosetting 10 h, T lympho

69

h, T lympho

119

h, T lympho

120

h, T lympho

130

h, T lympho

NKandother cytotoxiccell activities increased 51Cr 70 h, PBMNC release, K562 target cells

fiEND, non-opioids

fiEND, MENK

MENK MOR

fiEND > MENK

Annual Reviews OPIOIDS IN THE IMMUNE SYSTEM 229

Annu. Rev. Immunol. 1988.6:219-249. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Dose-response relationship

Stereospecificity

Further charactedzation

Antagonism

yes

NR

yes, NAL

BEND ineffective!

bell-shaped

NR

NR

des-Tyr ~zENDnot effective!

yes

NR

claimed, NAL (no data)

no

NR

no, NAL

bell-shaped

NR

NR

enhancedproliferation of PHA-stimulated PBL;reversal of PGE2inhibition BEND inhibits only slightly andonly under restricted conditions; lympho markersevaluated effects at 1-I00 nM BEND

NR

NR

NR

Con A and/~ENDare additive

bell-shaped

NR

no, NAL

effects as lowas 10 fM

yes, normal

yes, NAL

MOR decreased, MENK increased

yes

yes, dextrovs. levomoramide NR

yes, NAL

NR

unclear

NR

NR

NR

NR

no (?), NAL

NR

NR

NR

enhancedrosetting, claimed at <0.01 fM! Zn + MENK enhance rosetting effect on receptor "microdisplacement" in T-cell membrane

yes, normal

NR

yes, NAL

increase in numberof NKcells; LENK,MOR, ~ENDno effect

Annual Reviews 230

SIBINGA & GOLDSTEIN

Table2 (continued)

Annu. Rev. Immunol. 1988.6:219-249. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

References

Species, cell type

Effect, assay

NKandother cytotoxiccell activities (continued) 71 h, PBMNC increased 5~Cr release, K562 target cells ADCC assay also 72 increased5~Cr h, PBMNC release, K562 target cells ADCC assay also 73 h, PBMNC increased~tCr release, K562 target cells ADCC assay also 74 h, large increased5~Cr granular release, K562 lympho target cells ADCC assay also 75 decreased h, PBMNC release, K562 target cells; ADCC assay; monoclonal staining,T-cell subsets 76 h, PBL ~Crrelease, K562 target cells 77 r, spleno decreased5~Cr release, m lymphoma cells r, spleno decreased5~Cr 78 release, mlymphoma cells 131

m, spleen

Systemicin vivoeffects 79 m

i32 133

m, rabbits M~, PMNL h, PMNL, PBMNC

Agonist(s)and pattern of agonist potencies

fiENDderivative

),END> fi-lipotropin fiEND > ~tEND= LENK > MOR MENK = LENK

fiEND > trEND = yEND

fiEND = DYN = MENK

fiEND MOR;stress

MOR;stress

increased5~Cr release, m mastoeytomaor thymomacells

MENK,fiEND

inhibition of primary immuneresponse, increasedspleen/ bodywt ratio resistanceto infection multipleassays

MOR

MOR MOR,methadone

Annual Reviews OPIOIDS

Annu. Rev. Immunol. 1988.6:219-249. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Dose-response relationship

Stereospecificity

IN THE IMMUNE SYSTEM

Antagonism

231

Further characterization

NR

NR

NR

enhanced NK activity but not ADCC

bell-shaped

NR

yes, NAL

NALvs. interferon effects

bell-shaped

NR

NR

yes, normal

NR

yes, NAL

supernatants of PBMNC incubated with opioid enhance NKactivity /~ENDalso blocks IFN-~, production

no

NR

variable,

unclear

NR

yes, NAL

yes, normal

NR

yes, NAL

NR

NR

bell-shaped

yes, LEV/DEX (cited abstract) NR

partial

peak effects with BEND 100 pM, MENK 1 pM(!) effector cells Lyt2+ or Thy-1 +

yes

NR

yes, NAL

chronic MORto 400 mg/kg daily

yes

NR

no,

NR

NR

multiple immunosuppressive effects multiple immunosuppressive effects

NAL

NAL

no change in T-cell markers with opioid preincubation

large between-subjects variability in response MORor "opioid" type stress suppress NKactivity tolerance to MOR,no cross-tolerance to stress

Annual Reviews

232

SIBINGA & GOLDSTEIN

Table2 (continued)

References

Species, cell type

Effect, assay

Agonist(s) and pattern of agonist potencies

Annu. Rev. Immunol. 1988.6:219-249. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Systemicin vivoeffects (continued) 80

h, PBL

decreased numberof T cells

unclear, opiate addicts

134

h, PBL

stimulation of leuko inhibitory factor production

fiEND

81

number of pulmonary metastases tumor growth enhanced by stress

MOR,pentazocine

82

r, Walker256 cells r, tumors

83

r, spleno

icv MOR

84

r, tail-flick assay

85

r, body temperature

86

r, splenic mononuclear cells

decreased NKactivity by 51Crrelease assay decreasedeffect of MORafter cyclosporineor irradiation IFN~prevents tolerance to MOR hypothermia cyclosporineprevents MORwithdrawal syndrome

121

h, AIDS patients h, normal volunteers

changesin T-cell subsets increasedT-cell rosette formation

MENK10/~g/kg

135

MOR

MOR

MOR NA

MENK

Stereospecificity In functional assays this can be demonstratedby using an agonist and its enantiomer--e.g, levorphanol (LEV)and dextrorphan (DEX)or antagonist and its enantiomer [e.g. (-)NALand (+)NAL]. Antagonism Antagonismby NAL(or the closely related naltrexone--NTX) is essential characteristic of opioid-mediated effects. NAL has greatest affinity for/~ receptors, but at higher concentrationswill also block the other

Annual Reviews OPIOIDS IN THE IMMUNESYSTEM 233

Annu. Rev. Immunol. 1988.6:219-249. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Dose-response relationship

Stereospecificity

Further characterization

Antagonism

NA

NR

NR

NR

yes, NALon cells, rosetfing NR

"immunecompetence" decreasedin addicts

NR

NR

yes, NAL

NA

NR

claimed, NTX

yes

NR

yes, NTX

NR

NR

NR

I00 pMenhances production of leukocyte migration inhibitory factor MOR increases number of metastases footshock or MOR enhanced tumor growth, NTXblocked N-methyl MORhad noeffect given systemically NR

NR

NR

NR

NR

NA

NA

NA

single dose

NR

NR

effect can be transferred by splenic mononuclear cells NR

variable

NR

NR

effects at subnanomolar concentrations

opioid receptor types. Differentiation of receptor types in a mixedpopulation is possible, in principle, with highly selective antagonists, but the availability of these for opioid receptors is still limited. ICI 174864has goodselectivity but only modestaffinity for fi receptors (53). (-)Mr2266 has fairly high affinity but poorselectivity for x over/~(54). Antagonism dependson the affinities of both agonist and antagonist for the receptor, and on the concentrations of both (55). Antagonism must be assessed with concentrations of an antagonist well aboveits ownreceptor dissociation constant, even whenthe agonist concentration is very low. With highaffinity agonists, usinga molarequivalentconcentrationor arbitrary excess

Annual Reviews 234

SIBINGA & GOLDSTEIN

of antagonist (as is often done)lacks any theoretical basis and maywell fail to demonstratethe antagonism. As an example, suppose the Kd of NALis 2 nMand an agonist is effective at 20 pM. Despite the low concentration of agonist, at least 2 nMNALwill be required to occupy half the receptors.

Annu. Rev. Immunol. 1988.6:219-249. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Further Characterization Derivatives of opioids can be used to establish the opioid nature of an observedpharmacologiceffect. Thus, the sameconsequencesof structural modificationapply here as wcrcdiscussedearlier for binding. Theabsolute concentrationof an opioid required for a functional effect is importantin assessing the possible physiologic relevance in relation to the probable concentrationsof endogenous opioids at target sites in vivo. Wehavedivided the functional studies into sevencategories: (a) chcmotaxis, (b) macrophagc (M~)and granulocytc(PMNL) activity, (c) on mastcells, (d) lymphocyte proliferation and function, (e) T cell rosctting, (f) NKandother cytotoxiccell activities, (#) systemicin vivoeffects. Inasmuchas blockade or reversal by NAL(or equivalent antagonist) essential to the definition of a functionalopioid-mcdiatcd effect, wcusually discuss only results that meet this criterion; however,data fromall the papers are summarizedin Table 2. CHEMOTAX~S VanEpps&Saland (56) first reported a chcmotacticeffect fiENDand MENK on humanmonocytes. Cells migrated into a membrane towardthe chambercontaining the opioid peptidc at concentrationsin the picomolarrange; no such directional migrationwasseen if both chambers contained the peptidc. The dose-response curve was curiously bimodal, with peak effects at about 1 PMand 10 riM. NAL~at l0 nMalmost totally abolished the effect. Monocytesfrom only about 7~%of subjects showed these chcmotacticresponses; ncutrophils fromall subjects were muchless responsive.Infusion of fiENDby the icv route in rats led to migrationof M~b-likecells throughthe ependymal lining of the ventricles; unfortunately, NALblockadeof this interesting phenomenon was not reported. Ruff et al (57) confirmedthe in vitro findings. Theyshowedthat the positive chemotaxiswasstereospecific; the enantiomericagonist pair levallorphan/dextrallorphanwas used, as well as the antagonist pair (--)NAL/ (+)NAL.Most remarkable, DADLE was effective at concentrations low as 10 fM, with maximum effect at about 10 pM.The order of agonist potencies was fiEND > DADLE= DYN> LEV > BREM,and MOR waswithout effect. This pattern suggests that e receptors maybe responsible. Simpkinset al (58) found that fiENDslightly enhancedthe chemotaxis

Annual Reviews

Annu. Rev. Immunol. 1988.6:219-249. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

OPIOIDS

IN THE IMMUNE SYSTEM

235

of PMNLtoward the attractant fMLPin a soft agar medium. The fiEND dose-response curve was bell-shaped, with maximum effect at about 1 nM. In cells from eight subjects, NALat 1/~Mantagonized the effect. However, the assay was not robust; the effect itself was small (about 20%increase in migration) and showedconsiderable intersubject variability. Falke & Fischer (59) depicted changes in PMNLmorphology caused by fiEND or LEV;these effects were stereospecific and could be antagonized by DIP. They were qualitatively similar to the cell elongation caused by fLMP. The authors speculate that such morphologic effects might be related to the cell adherenceand motility that are relevant to the physiologic role of PMNL. Horvfith et al (60) found that an inhibitory effect of the wheat protein ,-gliadin on humanleukocyte migration was antagonized by NAL,as was the similar effect of MOR,whereas inhibition by PHAwas unaffected by NAL.The interest of this finding lies in the sensitivity of patients with celiac disease to a-gliadin, and the fact that wheat gluten is a source of opioid peptides (61). AND GRANULOCYTE(PMNL) ACTIVITY F6ris et al (62, 63) reported that preincubating rat peritoneal M~bwith MENK caused enhanced lysis of antibody-coated target cells (SRBC). The effect was observed in the range 1-100 nMbut not at higher concentrations, and it was blocked almost completely by NALat 1 #M. A complex of other phenomena was reported, such as enhanced superoxide production, increased Na÷ uptake at 1 nM MENK,and increased Ca2+ uptake at 1 #MMENK.NALblockade was not reported for an.y of these effects. Cyclic nucleotide changes reflected the distinction between low- and highconcentration effects in that cGMPwas more than doubled at l0 nM MENK,with return to normal levels at higher concentrations, whereas cAMPincreased only at concentrations in the micromolar range. NAL(at the very high concentration of l0 #M) blocked the MENK effect on cGMP but not on cAMP.The authors attempt to bring all these complicated and diverse observations together, probably prematurely. It seems clear enough that there are opioid-mediated effects worthy of study at low concentrations of MENK.Especially interesting would be a decisive study with NAL,including a proper determination of the NALaffinity (Ko) for the responsible receptors (55). The authors’ suggestion that ~ receptors mediate the observed effects on M~bmust be regarded as merely speculative at present. Sharp et al (64) also demonstrated enhanced superoxide production both DYNand fiEND; the effect on M~blasted only a few minutes, while that on PMNL (shown in the same experiments) was longer lasting. With MACROPHAGE(M~b)

Annual Reviews 236

SIBINGA& GOLDSTEIN

Annu. Rev. Immunol. 1988.6:219-249. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

PMNLthe effects occurred at 1-100 pMbut not at much higher concentrations (bell-shaped dose-response curve), whereasa robust effect was seen on M~bonly at about 1/~M. Brief pretreatment of the cells with 10 #M (sic!) NALsubstantially reduced the effects of DYN,fiEND, and MOR,but no controls with NALalone or with (+)NALwere reported. EFFECTS ONMAST CELLSYamasakiet al (65) reported that the inhibition of IgE-stimulated release of serotonin fro.m rat mast cells by isoproterenol, PGE~,or dopamine was prevented by 0pioids in a stereospecific and NALreversible manner. LEVwas effective at 100 nM, DEXwas ineffective, and MORrequired 5 #M. The evidence points clearly to a non-# opioid receptor mediated effect, but unfortunately, no panel of agonists and antagonists was studied. Liu et al (66) found a stereospecific and NAL-blocked increase calcium binding in rat peritoneal mast cells. Agonist potencies were //END > LEV> MOR.The site of binding was shown to be on the plasma membrane, suggesting an effect on calcium entry that might mediate degranulation. Curiously, however, these investigators could not find any effect of MORon histamine release in concentrations up to 0.5 mM,a finding they found "perplexing" in view of the fact that MORincreases plasmahistamine levels. It maybe that the rat is exceptionally resistant to this effect. Opioids administered intradermally elicited a wheal-and-flare response in humanskin (67); evidence of mast cell degranulation was adduced electron microscope studies on biopsy specimens. The order of agonist potencies (see Table 2) suggests that g and/or, receptors maybe responsible. The activity of codeine is anomalousunless a demethylase activity converted the codeine to MOR,inasmuch as codeine itself is virtually unrecognized by opioid receptors. NALblocked the MOReffect but not that of DYN.However,only a single (possibly insufficient) NALdose was tested. On the whole, the evidence is supportive of an opioid-mediated action. LYMPHOCYTE PROLIFERATION ANDFUNCTION Johnson et al (42) reported that sEND, MENK,and LENK(with approximately equal potency, 50 nM) decreased the number of mouse splenocytes producing antibody in plaque-forming assays. The effect was blocked by NAL.Curiously,//END was inactive, suggesting that the receptors maybe of the 3 type. Heijnen et al (68) confirmed these re,suits, and although NALantagonism was not tested, they showed elegantly that [des-Tyrl]flEND was inactive. This strongly implicates an opioid receptor mediation of the effect, even though NALblockade was not studied.

Annual Reviews OPIOIDS IN THE IMMUNESYSTEM237

Annu. Rev. Immunol. 1988.6:219-249. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Farrar (40) found that/~END caused nearly a twofold stimulation thymidine incorporation into PHA-stimulated IL-2 dependent human T lymphocytes from peripheral blood. The effect was dose-dependent and was maximal at subnanomolar concentrations. ]~-ENDalso reversed the inhibition of proliferative responses caused by PGE2.This latter effect was stated to be blocked by NAL,but no data were given. T CELLROSETTING Several studies have confirmed the initial report of Wybranet al (10) showing that preincubation with 1 nMMORdecreased the numberof active (i.e. high-affinity) rosettes formedby humanperipheral blood lymphocytes on brief exposure to SRBC.The effect was doserelated and was blocked by NAL.MENK had no effect at a comparable concentration but in the micromolar range increased the number of rosettes, also in a NAL-reversible manner. De Carolis et al (69) found that ]~END(active in suppressing rosetting at 10 pM) was more potent than MOR,and they failed to observe an increase with MENK and LENK.All agonist effects were NAL-reversible. NKANDOTI-I~R CYTOTOXIC CELLACTIVITYMathews et al (70) reported NAL-reversible enhancement of the natural cytotoxicity of human PBMNC using a standard 51Cr lysis method with K562 tumor cells as targets./~END was effective at concentrations as low as 10 fM, MENK at 10 pM. Enhancementwas shownat all effector: target cell ratios and was further shownto represent increased recruitment of effector cells from the pre-NK population as well as accelerated cytotoxicity. LENK,~END, and MORwere ineffective. In a follow-up study (71) with a/~ENDderivative more resistant to degradative enzymes, the earlier results were confirmed, and no opioid effects on ADCCwere observed. Kay et al (72) found a similar enhancement of NKfunction but with quite different dose relationships./~END was ineffective below 1 nMand above 100 nM, yielding a bell-shaped dose-response curve. NALonly partially blocked the effect even at the (very high) 10/~Mconcentration tested. Interestingly,/~-lipotropin was as potent as ]~END;if the effect is on opioid receptors, this result implies that the peptide was cleaved to yield ]~END,since/~-lipotropin itself does not bind to opioid receptors. Enhancement of NKactivity by human IFN was found to be blocked by NAL,but the source and purity of the IFN preparation is not stated, so no interpretation is possible. In an attempt to study the mechanism, Wybran(73) showed that the enhancing effect of MENK or LENKon NKactivity was manifested in the supernatant after incubation with PBMNC, for when this superuatant was used in an assay with cells of another donor, their NKactivity was

Annual Reviews

Annu. Rev. Immunol. 1988.6:219-249. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

238

SIBINGA & GOLDSTEIN

enhanced.Asin all NKassays, the observedenhancements are small (here about 10%),so decisive interpretations are difficult, but the data seemto indicatethat the opioideffect is on the effectorcell, not onthe target cells. Similar results were found by Mandleret al (74), using/~END at 10100 pMand with clear evidence of blockade by 100 nMNAL.They also measured a concomitant augmentation of IFN production by/~END. In contrast to the above,Prete et al (75) claimedto showa reduction NKactivity by/~END,DYN,and MENK, blocked by NAL,but the data are not convincing. DeSanctis et al (76) observeda difference betweenperipheral blood lymphocytes(PBL)from different donorsin that those with low baseline NKactivity showed enhancementwith/~END,whereas those with high initial activity tended to showa reduction. Botheffects were blockedby NAL.However,no clear dose-response relationship emerged, nor was analysis of variance presented to showthat within-donor and betweendonorvariances werereally different. Shavit et al (77, 78) found that NKactivity of splenocytes from rats subjected to a certain pattern of stressful footshockwasdecreased, compared with controls. This group has maderemarkableobservations on the differences in analgesia producedby various kinds of footshock--some NAL-reversible,somenot--and found that the depression of NKactivity by footshockcorrelates with these differences. Althoughchronic administration of MOR led to tolerance to the effect on NKactivity, the footshock-inducedeffect on NKactivity did not becomecross-tolerant to MOR. svs~mc(rN XaVO)ErrEC~Giing6r et al (79) reported a NAL-reversible depression of spleen:body weight ratio and serum hemolysintiter in MOR-treated mice immunized with SRBC. McDonough et al (80) studied the numbersof B and T lymphocytes peripheral bloodof street addicts primarily addictedto heroin but also in populations maintained on methadone.Use of marihuanawas determined by questionnaire.Thepercentageof B cells wasnot different fromcontrol, but that of T cells (determinedby rosetting) was only 23 and 32 in two addict populations, as comparedwith 67 and 71 in control populations. The reported use or non-use of marihuana had no influence on these frequencies. Interestingly, incubationof the cells with 10-t00nMnaloxone (or alternatively, with dibutyryl cyclic AMP) reversed the depressionof cell frequencyat the expenseof the null class of lymphocytes,as though the chronic opiate exposurehad preventedexpression of the cell-surface receptors responsible for rosetting. As with so manystudies on human

Annual Reviews

Annu. Rev. Immunol. 1988.6:219-249. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

OPIOIDS IN THE IMMUNESYSTEM239 populations, this one suffers from the serious defect that the "normal" controls may not be entirely comparable with the subject group. Further evidence of an effect of MORon tumor growth is provided in a study by Simon & Arbo (81), who found that pulmonary metastases Walker 256 tumors in rats were increased by MOR,but not after NAL pretreatment. Shavit et al (82) showed not only that NKcytotoxic activity was suppressed in animals that received chronic footshock stress (see preceding section), but also that the growth of implanted tumors was enhanced. Both these effects were mimicked by MORand blocked by NAL.Surprisingly, the suppressant action of MORon NKactivity was found to be centrally mediated (83) in that effective doses given into the cerebral ventricles were a thousand times smaller than those required systemically. Moreover, a MOR analogue that does not cross the blood-brain barrier was ineffective. Dougherty et al (84, 85) have linked systemic treatments affecting immunefunction to changes in MOR effects in the rat--decreased antinociceptive effect in rats pretreated with cyclosporine or gammaradiation, and suppressed tolerance to MOR-inducedhypothermia after IFN~ treatment. Most recently, the same group reported (86) that cyclosporine attenuates the opiate withdrawal syndromein rats, and furthermore, that this effect can be transferred passively by splenic mononuclear cells from cyclosporine-treated animals. Inasmuch as at least some of the opiate withdrawal disturbances are centrally mediated, these, findings raise provocative questions about immunesystem influences on brain function. Finally, although their relationship to the subject of this review may be remote, the remarkable findings of the De Wied group (87, 88) schizophrenia and HLAantigens merit comment. Some schizophrenic patients had been found to respond favorably to treatment with the/~END fragment [des-Tyr]~-endorphin (DT~E).This peptide, about half the length of/~END,is devoid of opioid activity because tyrosine-1 is missing; so nothing under discussion here concerns opioid receptors. A correlation was found between the presence of certain HLAclass I antigens and a favorable response to DT~E.Thus, the relative likelihood of improvement was 3-5 times greater in patients with B13, B15, or B22than in those with other HLAclass I antigens. The authors speculate whether schizophrenia is an autoimmunedisease and what possible ways the HLAantigens might be involved. Summary It is increasinglyclear that there is a variety of direct opioid effects on cells of the immunesystem, probably mediated by all four opioid receptor

Annual Reviews

240

SIBINGA & GOLDSTEIN

Annu. Rev. Immunol. 1988.6:219-249. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

types. Takingnaloxoneblockadeas the sine qua nonof an opioid receptormediatedeffect, wejudge the evidenceto be satisfactory for the following: (a) a chemotacdc action toward PMNC; (b) increased superoxide production by MS; (c) on mastcells, an increased bindingof calciumto plasmamembranes, prevention of the inhibitory effects of various agents on IgE-stimulated release of serotonin, and production of a wheal-and-flare response on intradermalinjection; (d) decreased antibody production by lymphocytes; (e) increased NKactivity. Numerouseffects on immunesystem function have also been reported after systemic administration of opioids, someof these opposite to the directly demonstratedin vitro effects. Thesemaywell be of physiologicor pathophysiologicimportance, but mechanismsremain obscure, rendering interpretations speculative. Indeed,evenfor the direct effects noted above, it should be borrte in mindthat the experimentsare pharmacologic,so that no single physiologicfunction can yet be adducedwith confidence. OPIOID

PEPTIDE

PRODUCTION

Ourcriteria/’or evaluatingstudies of opioid peptide productionin cells of the immunesystem focus on the specificity of the techniques employed. Molecularstudies with nucleic acid probes are potentially unambiguous in detecting mRNA, but there is no necessary correlation betweenlevels of mRNA and peptide content (89, 90). Noone has yet isolated an opioid peptide from cells of the immunesystem, and lack of rigorous antibody characterization often detracts from reports of opioid-like immunoreactivity. Preadsorptionto rule out unrelatedcross-reactivity is a requisite. In addition, since similar epitopes maybe sharedby unrelated polypeptides, the use of second (or even third) antibodies directed toward different epitopes on the moleculeof interest is highly desirable. The peptides themselves, after extraction and purification, mustbe analyzed to assess their identities. Opioidpepfides are knownto be processedin a variety of tissue-specific ways(and region-specific waysin the nervous system). Different processed forms mayhave very different biologic potencies--for example,N-acetylated fiENDcan not interact with opioid receptors, yet it reacts fully with most fiENDantibodies (91, 92)--so determinationof the precise molecularformof an opioid peptide is important in evaluatingits possiblephysiologicactivity. Table 3 summarizes the relevant studies. Zurawskiet al (93) recently reported the sequence of a cDNA clone

Annu. Rev. Immunol. 1988.6:219-249. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Annual Reviews OPIOIDS IN THE IMMUNE SYSTEM 241

Annual Reviews

Annu. Rev. Immunol. 1988.6:219-249. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

242 smr~A & GOLDSTEIN from a ConA-stimulated mouseT helper cell line, and they found an open reading frame with 93 %homologyto rat brain preproenkephalinmRNA. This sequence was abundant (0.1%-0.5%)in eDNA libraries generated fromfour inducedT helper lines, but only after mitogenstimulation. Cells that produced the mRNA also secreted ir-MENK. Consistentwith this finding, Monstein et al (94), usingprobesspecific for preproenkephalinmRNA and antibodies specific for enkephalin peptides, observedpositive results in leukocytesfromleukemiapatients. Lolait et al (95), using a eDNA probe, detected ACTH/fiEND message in mousespleen M~b.Furthermore, they showedby a combinationof RIA and HPLCthat the principal fiENDtranslation product in these cells had the properties of opioid-active (unacetylated) fiEND.In addition, significant amountof N-acetylatedfiENDwasalso present, as in pituitary. Extensiveanalyses by the samegroup (96) demonstratedir-fiENDand iro ACTH in extracts of adherent mousespleen cells, and they showedby gel permeationchromatography that the sizes were consistent with fiENDand ACTH (3500 and 4500daltons, respectively). Indirect immunofluorescence using these sameantisera showedir-fiENDin 12-19%of adherent cells, and all cells staining for fiENDwere also positive for the M~bmarker Mac-1.Preadsorption of the antiserum with fiENDabolished staining. A secondfiENDantiserumthat recognizeda different epitope yielded similar staining. Felten et al (97) found ir-MENK in rat splenic white pulp. Smithet (98) reported ir-ACTH and bioactivity as well as ir-fiENDin supernatants of humanPBLtreated with corticotropin releasing factor. Grube(99), using an antiserum raised against a NH2-terminalfragment of fiEND, namely ~END,found positive staining in a subpopulation of plasma cells in canine colonic mucosa.Preadsorptionwith fiENDabolished this staining. Findings by Blalock & Smith (100-102) linking opioid-like and ACTHlike immunoreactivity to IFNpreparations from virus-infected human leukocytes were held to reflect a structural relatedness betweenopioid peptides, ACTH, and IFN. However,no IFN sequences are significantly homologousto any opioid peptides or ACTH, and the proposedrelationship was also not borne out in studies using recombinantleukocyte IFN (103). Nevertheless,the possibility of cross-reactingepitopes in IFN~and the ACTH/fiEND precursor has been suggested (104, 105). Summary Of the three opioid genes, two havebeenshownto be expressedin cells of the immunesystem. Activation of T-helper lymphocytesby lectins or

Annual Reviews OPIOIDS

IN THE IMMUNE SYSTEM

243

antigens leads to production of preproenkephalin mRNA and secretion of enkephalinpeptides. Splenic lymphocyteshave similarly been shownto produce fiENDand ACTHand the corresponding mRNA.There are no reports yet of attemptsto detect expressionof the dynorphingene.

Annu. Rev. Immunol. 1988.6:219-249. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

CONCLUSION Opioid binding in cells of the immunesystem is at present poorly characterized, but there is strong evidencefor certain functionalopioideffects and for the productionof opioid peptides. Thesignificance of opioids in immunesystem function remains a matter for speculation, as there are multiple possible sourcesand targets. Opioidpeptides acting on receptors in cells of the immunesystem could comefrom within the system, from other peripheral opioid-producing tissues (e.g. adrenal medulla,pituitary) or, as has beensuggested(14, 106, 107), fromthe nervoussystem.Opioids producedwithin the immunesystem might act on autoreceptors, on other cells within the system,on local targets such as endothelial cells, or on neuronsof the central or peripheral nervoussystem. Non-opioidproducts of the immune system mightalso modulatethe actions of the opioids, as has beensuggestedfor IL-1 (108). Levelsof active circulating opioidpeptidesare difficult to measure(109112) and do not alone constitute strong evidencefor humorallymediated opioid regulation of or by the immune system. Circulating concentrations neednot reflect opioidlevels in the architecturally specializedareas where lymphocytematuration takes place in thymus, lymphnodes, and spleen. Preferential uptake, metabolism,andrelease of opioid peptides couldaffect local concentrations. Moreover, manyregulatory processes in immune function are thoughtto occur in discrete cell-to-cell interactions at short range(113), interactions that mightalso mediateopioideffects. Thepicture is complicatedgreatly by a confusion in the literature betweenthe opioid and non-opioid (i.e. not blocked by NAL)effects the opioid peptides. For example,such non-opioidinteractions of fiEND with lymphocytes through its COOH-terminaldomain have been well described(114). Schweigereret al (115, 116) foundnon-opioidbinding fiENDto a componentof humancomplement.Gilmanet al (117) reported enhancementand McCainet al (118) suppression of lymphocyte proliferation by fiENDthrough non-opioid interactions. Numerousother effects not reversible by NAL have beenreported (119-121). To gain a better understanding of the actions of endogenousopioid peptides on opioid receptors in the immunesystem, the starting points shouldbe phenomena that are robust, dose-related, andstereospecific, and

Annual Reviews

Annu. Rev. Immunol. 1988.6:219-249. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

244

srar~cA & C, OLt)ST~N

that are blocked or reversed by NAL.Then opioid binding should be studied in cell types and underconditions already established as meeting thesecriteria. For those trying nowto define the role of opioids in the immune system, establishedopioid science is rich in rigorous methodology for dealing with the complexities.For receptor study, agonists and antagonists with better stability and improvedreceptor type selectivity haveb~endeveloped,as havemethodsof type-selective protection and inactivation (25). Theavailability of opioid polynucleotide probes makesmolecularstudies in the immune system(as by in situ hybridization) possible. Methodsfor detailed analysis of processedforms of opioid peptides have also been perfected. Numerousspecific polyclonal and monoclonal antibodies have been developed and characterized. The understanding and capability gained in the study of opioids in the nervous system could be applied more systematically than heretofore to the study of opioids in the immune system. ACKNOWLEDGMENTS

This work was supported, in part, by grants AI07757from the National Institutes of Health (H. McDevitt)and DA-1199 from the National Institute on DrugAbuse(A. Goldstein). Literature Cited 1. Nakanishi,S., Inoue,A., Kita, T, Nakation of diversity of opioid peptides. amura, M., Chang, A. C. Y., Cohen, Biochem.Act. Hormones12:1 S. N., Numa, S. 1979. Nucleotide 6. Robson,L. E., Paterson,S. J., Kostersequence of cloned eDNAfor bovine litz, H. W.1983. Opiate receptors. corticotrophin,fl-lipotropinprecursor. HandbkPsychopharmacol.17:13 Nature 278:423 7. Snyder, S. H. 1984. Drug and neuro2. Noda, M.; Furutani, Y., Takahashi, transmitter receptorsin the brain. SciH., Toyosato,M., Hirose, T., Inayama, ence 224:22 8. Paterson, S. J., Robson,L. E., KosterS., Nakanishi,S., Numa,S. 1982. Cloning and sequence analysis of cDNA ritz, H. W.1984. Opioidreceptors. In for bovine adrenal preproenkephalin. The Peptides: Analysis, Synthesis, Biology, Vol. 6, ed. S. Udenfriend,J. Nature 295:202 Meienhofer,p. 147. Orlando,Fla: Aca3. Kakidani, H., Furutani, Y., Takahashi, H., Noda, M., Morimoto,Y., demic Hirose, T., Asai, M., Inayama,S., Nak9. Millan, M.J., Herz,A. 1985.Theendoanishi, S., Numa,S. 1982.Cloningand crinology of the opioids. Int. Rev. sequenceanalysis of eDNA for porcine Neurobiol.26:1 fl-neo-endorphin/dynorphinprecursor. I0. Wybran,J., Appelboom,T., Famaey, Nature 298:245 J.-P., Govaerts, A. 1979. Suggestive 4. Akil, H., Watson,S. J., Young,E., evidence for receptors for morphine and methionine-enkephalinon normal Lewis, M. E., Khachaturian, H., Walker, J. M. 1984. Endogenous humanblood T lymphocytes. J. Immuopioids: biology and function. Ann. nol. 123:1068 Rev. Neurosci.7:223 11. Fischer, E. G., Falke, N. E. 1984.flEndorphin modulates immunefunc5. Herbert,E., Civelli, O., Douglass,J., tions. Psychother.Psychosom.42:195 Martens, G., Rosen, H. 1985. Gener-

Annual Reviews

Annu. Rev. Immunol. 1988.6:219-249. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

OPIOIDS IN TI-tE 12. Editorial. 1984. Opiates, opioid peptides, and immunity.Lancet 1:774 system 13. Blalock, J. E. 1984. Theimmune as a sensory organ. J. ImmunoL132: 1067 14. Weber,R. J., Pert, C. B. 1984. Opiatergic modulation of the immune system. In Central and Peripheral Endorphins: BasicandClinical Aspects, ed. E. E. Miiller, A. R. Genazzani,p. 35. NewYork: Raven 15. Teschemacher, H., Schweigerer, L. 1985. Opioid peptides: do they have immunological Trends Pharmacol.Sci. significance? 6:368 16. Wybran, J. 1985. Enkephalins and endorphinsas modifiers of the immune system: present and future. Federation Proc. 44:92 17. Smith, E. M., Harbour-McMenamin, D., Blalock, J. E. 1985. Lymphocyte production of endorphins and endorphin-mediatcd immunoregulatoryactivity. J. Immunol. 135:779s 18. Blalock, J. E., Harbour-McMenamin, D., Smith, E. M. 1985. Peptide hormonesshared by the neuroendocrine and immunologicsystems. J. lmmunol. 135:858s 19. Morley,J. E., Kay,N., Allen, J., Moon, T., Billington, C. J. 1985.Endorphins, immune function, and cancer. Psychopharmacol.Bull. 21:485 20. Lewis, J. W., Shavit, Y., Terman, G. W., Nelson, L. R., Martin, F. C., Gale, R. P.0 Liebeskind,J. C. 1985.Involvement of opioid peptides in the analgesic, immunosuppressive, and tumor-enhancing effects of stress. Psychopharmacol.Bull. 21:479 21. Pert, C. B., Snyder,S. H. 1973.Opiate receptor: Demonstration in nervous tissue. Science179:1011 22. Simon,E. J., Hiller, J. M., Edelman, I. 1973. Stereospecificbinding of the potent narcotic analgesic [3H]etorphine to rat-brain homogenate.Proc. Natl. Acad. Sci. USA70:1947 23. Terenius,L. 1973.Stereospecificinteraction betweennarcotic analgesics and a synaptic plasma membranefraction of rat cerebral cortex. Acta pharmacol. et toxicol. (Kbh.)32:317 24. Lord, J. A. H., Waterfield, A. A., Hughes,J., Kosterlitz, H. W.1977. Endogenousopioid peptides: multiple agonists and receptors. Nature267:495 25. James,I. F., Goldstein,A. 1984.Sitedirected alkylation of multiple opioid receptors. I. Bindingselectivity. Mol. Pharmacol.25:337 26. Kosterlitz, H. W.1985.Opioidpeptides

IMMUNESYSTEM245

andtheir receptors. Proc.R. Soc. London Ser. B 225:27 27. Dixon, W. R., Viveros, O. H., Unsworth, C. D., Diliberto, E. J., Way, E. L., Lewis,J. W.,Chandra,A., Holaday, J. W.,Long,J. B., Tortella, F. C. 1985.Multipleopiate receptors: functional implications. FederationProc. 44:2851 28. Goldstein, A., Lowney,L. I., Pal, B. K. 1971.Stereospecificandnonspecific interactiohs of the morphineco~agener levorphanolin subcellular fractions of mousebrain. Proc. NatL Acad. Sci. USA 68:1742 29. Klee, W.A., Nirenberg, M. 1974. A neuroblastoma X glioma hybrid cell line with morphinereceptors. Proc. Natl. Acad. Sci. USA71:3474 30. Htllt, V., Wiister, M.1978.Theopiate receptors. In Developmentsin Opiate Research,Ch. 1, ed. A. Herz, p. 1. New York/Basel: Marcel Dekker 31. Lord,J. A. H., Rance,M.J., Smith,C. F. C. 1982. Assayof the endorphins and related analogs. In Endorphins: Chemistry, Physiology, Pharmacology, andClinicalRelevance,ed. J. B. Malick, R. M.S. Bell, p. 57. NewYork/Basel: Marcel Dekker 32. Goldstein, A., James,I. F. 1984.Multiple opioid receptors: criteria for identificationandclassification. Trends Pharrnacol.Sci. 5:503 33. Beddell,C. R., Clark, R. B., Hardy,G. W., Lowe,L. A., Ubatuba,F. B.,Vane, J. R., Wilkinson, S., Chang, K.-J., Cuatrecasas, P., Miller, R. J. 1977. Structural requirementsfor opioid activity of analoguesof the enkephalins. Proc. R. Soc. LondonSet. B 198:249 34. Schiller, P. W.1984. Conformational analysis of enkephalin and conformation-activity relationships. In The Peptides:Analysis, Synthesis,Biology, Vol. 6, ed. S. Udenfriend, J. Meienhofer, p. 219. Orlando,Fla: Academic 35. Pert, C. B., Pasternak,G., Snyder,S. H. 1973. Opiate. agonists and antagonists discriminated by receptor binding in brain. Science 182:1359 36. Blume, A. J. 1978. Interaction of ligands with the opiate receptors of brain membranes:Regulation by ions and nucleotides. Proc. NatLAcad.Sci. USA75:1713 37. Lopker, A., Abood,L. G., Hoss, W., Lionetti, F. J. 1980. Stereoselective muscarinic acety~choline and opiate receptors in humanphagocytic leukocytes. Biochem.Pharmacol.29:1361 38. Mehrishi, J. N., Mills, I. H. 1983.

Annual Reviews

Annu. Rev. Immunol. 1988.6:219-249. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

246

SIBINGA & GOLDSTEIN

Opiate receptors on lymphocytesand platelets in man.Clin. Immunol.Immunopathol.27:240 39. Ausiello,C. M., Roda,L. G. 1984.Leuenkephalin binding to cultured human T lymphocytes.Cell Biol. Int. Rep. 8: 97 40. Farrar, W. L. 1984. Endorphinmodulation oflymphokine activity. In Opioid Peptidesin the Periphery,ed. F. Fraioli, A. Isidori, M. Mazzetti, p. 159. Amsterdam/NewYork/Oxford: Elsevier Sci. 41. Falke, N. E., Fischer, E. G., Martin, R. 1985. Stereospecificopiate binding in living humanpolymorphonuclear leucocytes.Cell BioLInt. Rep. 9:1041 42. Johnson,H. M., Smith, E. M., Torres, B. A., Blalock, J. E. 1982. Regulation of the in vitro antibody response by neuroendocrinehormones.Proc. Natl. Acad. Sci. USA79:4171 43. Falke, N. E., Fischer, E. G. 1986.Opiate receptormediatedinternalization of ~25I-fl-endorphin in humanpolymorphonuclearleucocytes. Cell Biol. Int. Rep. 10:429 44. Mendelsohn,L. G., Kerchner, G. A., Culwell, M., Ades, E. W. 1985. Immunoregulationby opioid peptides: I. Absenceof classical opioid receptor on humanmononuclear cells. J. Clin. Lab. Immunol. 16:125 45. Mosberg, H. I., Omnaas, J. R., Goldstein,A. 1987. Structural requirementsfor delta opioid receptor binding. Mol. Pharmacol.31:599 46. Paul, W.E., Ohara~J. 1987. B cell stimulatoryfactor-i/intedeukin 4. Ann. Rev. Immunol.5:429 47. Dower,S. K., Urdal, D. L. 1987. The interleukin-1 receptor. Immunol.Today 8:46 48. Robb,R. J., Greene, W.C., Rusk, C. M.1984. Lowand highaffinity cellular receptors for interleukin 2. Implications for the level of Tacantigen. J. Exp. Med. 160:1126 49. Park, L. S., Friend, D., Gillis, S., Urdal, D. L. 1986. Characterization of the cell surface receptor for a multi-lineagecolony-stimulatingfactor (CSF-2c 0. J. Biol. Chem.261:205 50. Walker,F., Burgess, A. W.1985. Specific binding of radioiodinated granulocyte-macrophagecolony-stimulating factor to hemopoieticcells. EMBO J. 4:933 51. Goldstein,A., James,I. F. 1984.Sitedirected alkylation of multiple opioid receptors. II. Pharmacologicalselectivity. Mol. Pharmacol.25:343 52. Schulz, R., W/ister, M., Krenss, H.,

Herz, A. 1980. Sdective development of tolerance without dependencein multiple opiate receptors of mousevas deferens. Nature285:242 53. Cotton, R., Giles, M.G., Miller, L., Shaw, J. S., Timms, D. 1984. ICI 174864:a highly selective antagonist for the opioidh-receptor.Eur. J. Pharmacol. 97:331 J., Paterson,S. J., Tavani,A., 54. Magnan, Kosterlitz, H. W.1982. The binding spectrumof narcotic analgesic drugs with different agonist and antagonist properties. Naunyn-Schmiedebery’s Arch. PharmacoL319:197 55. Kosterlitz, H. W., Watt, A. J. 1968. Kinetic parametersof narcotic agonists andantagonists, with particular reference to N-allylnoroxymorphone(naloxone). Br. J. Pharmacol.Chemother. 33:266 56. VanEpps, D. E., Saland, L. 1984. flEndorphinand met-enkephalin stimulate humanperipheral blood mononuclear cell chemotaxis. J. Immunol. 132:3046 57. Ruff, M. R., Wahl, S. M., Mergenhagen, S., Pert, C. B. 1985. Opiate receptor-mediated chemotaxis of humanmonocytes. Neuropeptides 5:363 58. Simpkins,C. O., Dickey, C. A., Fink, M. P. 1984. Human neutrophil migration is enhancedby beta-endorphin. Life Sci. 34:2251 59. Falke, N. E., Fischer, E. G. 1985.Cell shape of polymorphonuclear leukocytes is influencedby opioids. Immunobiology 169:532 60. Horv~th,K., Grhf, L., Walcz,E., Bod~inszky,H., Schuler,D. 1985.Naloxone antagoniseseffect of ct-gliadin on leucocyte migrationin patients with coeliac disease. Lancet2:184 61. Zioudrou,C., Streaty, R. A., Klee, W. A. 1979. Opioidpeptides derived from food proteins. J. Biol. Chem.254:2446 62. Frris, G., Medgyesi,G. A.., Gyimesi, E., Hauck, M. 1984. Met-enkephalin induced alterations of macrophage functions. Mol. Immunol.21:747 63. Fbris, G., Medgyesi,G. A., Hauck,M. 1986.Bidirectionaleffect of met-enkephalin on macrophageeffector functions. Mol. Cell Biochem.69:127 64. Sharp, B. M., Keane, W.F., Sub, H. J., Gekker,G., Tsukayama, D., Peterson, P. K. 1985. Opioidpeptides rapidly stimulate superoxide production by humanpolymorphonuclear leukocytes and macrophages. Endocrinology 117:793 65. Yamasaki, Y., Shimamura,O., Kizu, A., Nakagawa,N., Ijichi, H. 1983.

Annual Reviews

Annu. Rev. Immunol. 1988.6:219-249. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

OPIOIDS IN THE IMMUNESYSTEM 247 Interactions of morphinewith PGE1, isoproterenol, dopamineand aminophylline in rat mastcells; their effect on IgE-mediated 14C-serotoninrelease. Agents Actions 13:21 66. Liu, J.-S., Garrett, K. M., Lin, S.-C. C., Way,E. L. 1983.Theeffects of opiates on calcium accumulation on rat peritoneal mastcells. Eur. J. Pharrnacol. 91:335 67. Casale, T. B., Bowman, S., Kaliner, M. 1984. Induction of humancutaneous mastcell degranulationby opiates and endogenousopioid peptides: evidence for opiate and nonopiatereceptor participation. J. AllergyClin. Immunol. 73: 775 68. Heijnen,C. J., Bevers, C., Kavelaars, A., Ballieux,R. E. 1986. Effect of ctendorphinon the antigen-inducedprimary antibody response of human bloodBcells in vitro. J. Immunol. 136: 213 69. De Carolis, C., De Sanctis, G., Perricone,R., Moretti, C., Fabbri, A., Gnessi, L., Fraioli, F., Fontana, L. 1984. Evidencefor an inhibitory role of fl-endorphin and other opioids on humantotal T rosette formation. Experientia 40:738 70. Mathews, P. M., Froelich, C. J., Sibbitt, W.L. Jr., Bankhurst,A. D. 1983. Enhancementof natural cytotoxicity by fl-endorphin. J. Immunol. 130:1658 71. Froelich, C. J., Bankhurst,A. D. 1984. Theeffect of fl-endorphin on natural cytotoxicity and antibody dependent cellular cytotoxicity.Life Sci. 35:261 72. Kay,N., Allen, J., Morley,J. E. 1984. Endorphins stimulate normal human peripheral blood lymphocytenatural killer activity. Life Sci. 35:53 73. Wybran, J. 1985. Enkephalins and endorphins: activation molecules for the immunesystemand natural killer activity? Neuropeptides 5:371 74. Mandler,R. N., Biddison, W.E., Mandler, R., Serrate, S. A.1986.fl-Endorphin augmentsthe cytolytic activity and interferon production of natural killer cells. J. Immunol. 136:934 75. Prete, P., Levin, E. R., Pedram,A. 1986.Thein vitro effects of endogenous opiateson naturalkiller cells, antigenspecificcytolyticT cells, andT-cellsubsets. Exp. Neurol.92:349 76. DeSanctis, G., DeCarolis, C., Moretti, C., Perrieone,R., Fabbri, A., Gnessi, L., Fraioli, F., Fontana, L. 1986. Endogenousopioids and the immune system:Demonstrationof inhibiting as well as enhancingeffects of fl-endor-

phin on natural killer activity. EOSRiv. di Immunol.ed Imrnunofarmacol. 6:13 77. Shavit, Y., Lewis, J. W., Terman,G. W.,Gale, R. P., Liebeskind,J. C. 1984. Opioid peptides mediate the suppressiveeffect of stress on naturalkiller cell cytotoxicity. Science223:188 78. Shavit, Y., Terman,G. W., Lewis, J. W., Zane, C. J., Gale, R. P., Liebeskind, J. C. 1986.Effects of footshock stress and morphineon natural killer lymphocytesin rats: studies of tolerance and cross-tolerance. Brain Res. 372:382 79. GiJng6r, M., Gent, E., Sa~duyu,H., Eroglu, L., Koyuncuo~lu, H. 1980. Effect of chronic administration of morphineon primary immuneresponse in mice. Experientia36:1309 80. McDonough,R. J., Madden, J. J., Falek, A., Sharer, D. A., Pline, M., Gordon,D., Bokos,P., Kuehnle,J. C., Mendelson,J. 1980. Alteration of T and null lymphocytefrequenciesin the peripheral blood of humanopiate addicts: in vivo evidence for opiate receptor sites on T lymphocytes.J. Immunol. 125:2539 81. Simon,R. H., Arbo, T. E. 1986. Morphine increases metastatic tumor growth.Brain Res. Bull. 16:363 82. Shavit, Y., Terman,G. W., Martin, F. C., Lewis, J. W., Liebeskind, J. C., Gale, R. P. 1985. Stress, opioid peptides, the immune system, and cancer. J. Immunol.135: 834s 83. Shavit, Y., Depaulis,A., Martin,F. C., Terman,G. W., Pechnick, R. N., Zane, C. J., Gale, R. P., Liebeskind,J. C. 1986. Involvement of brain opiate receptors in the immune-suppressive effect of morphine.Proc. Natl. Acad. Sci. USA83:7114 84. Dougherty, P. M., Aronowski, J., Samorajski,T., Dafny,N. 1986. Opiate antinociception is altered by immunemodification:the effect of interferon, cyclosporine and radiation-induced immunesuppression upon acute and long-term morphine activity. Brain Res. 385:401 85. Dougherty,P. M., Harper, C., Dafny, N.1986.Theeffect of alpha-interferon, cyclosporineA, and radiation-induced immunesuppression of morphineinduced hypothermia and tolerance. Life Sci. 39:2191 86. Dougherty, P. M., Aronowski, J., Drath, D., Dafny, N. 1987. Evidence of neuro-immunologicinteractions: Cyclosporine modifies opiate withdrawal by effects on the brain and

Annual Reviews

Annu. Rev. Immunol. 1988.6:219-249. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

248

SIBINGA & GOLDSTEIN immune components. J. Neuroimmunol. 13:331

endorphins. Nature 321:881 99. Grube, D. 1980. c~-Endorphin-like 87. de Jongh, B. M., Verhoeven,W.M. A., immunoreactivity in plasmacells of the van Ree, J. M., de Wied,D., van Rood, canine colonic mucosa.Histochemistry J. J. 1982. HLA,and the response to 69:157 treatment with y-type endorphins in 100. Blalock, J. E., Smith, E. M. 1980. schizophrenia. J. Immunogenetics9: Human leukocyteinterferon: structural 381 and biological relatedness to adreno88. Claas, F. H. J., van Ree, J. M., Vercorticotropic hormone and endorhoeven,W.M. A., van der Pod, J. J., phins. Proc. NatLAcad. Sci. USA77: Verduyn,W., de Wied,D., van Rood, 5972 J. J. 1986.Theinteraction between~- 101. Blalock, J. E., Smith, E. M. 1981. type endorphinsand HLAclass I antiHumanleukocyte interferon (HulFNgens. HumanImmunol. 15:347 c0: potent endorphin-like opioid activity. Biochem.Biophys.Res. Commun. 89. Howells, R. D., Kilpatrick, D. L., Bailey, L. C., Noe,M., Udenfriend,S. 101:472 1986. Proenkephalin mRNA in rat 102. Smith, E. M., Blalock, J. E. 1981. Humanlymphocyte production of heart. Proc. NatLAcad. Sci. USA83: 1960 c0rticotropin and endorphin-like sub90. Douglass, J., Cox, B., Quinn, B., stances: association with leukocyte interferon. Proc. Natl. Acad.Sci. USA Civelli, O., Herbert, E. 1987. Expression of the prodynorphingene in 78:7530 male and female mammalianrepro103. Wetzel,R., Levine, H. L., Hagman, J., duetive tissi~es. Endocrinology 120:707 Ramachandran,J. 1982. Humanleu91. Zakarian, S., Smyth,D. G. 1982. /~kocyte interferon has no structural Endorphinis processeddifferently in or biological relationship to corticospecific regions of rat pituitary and tropin. Biochem.Biophys.Res. Commun. brain. Nature296:250 104:944 92. Smyth, D. G. 1984. Chromatography 104. Jrrnvall, H., Persson, M., Ekman, of peptides related to /~-endorphin. R. 1982. Structural comparisons of leukocyte interferon and pro-opioAnal. Biochem.136:127 93. Zurawski, G., Benedik, M., Kamb,B. melanocortin correlated with immuJ., Abrams, J. S., Zurawski,S. M., Lee, nological similarities. FEBSLett. 137: F. D. 1986. Activation of mouseT153 helper cells induces abundantprepro- 105. Smith,E. M., Blalock, J. E. 1982. The enkephalin mRNA synthesis. Science hormonal nature of the interferon system. Tex. Rep. Biol. Med.41:350 232:772 94. Monstein,H.-J., Folkesson, R., Ter- 106. Pert, C. B., Ruff, M.R., Weber,R. J., enius, L. 1986. ProenkephalinA-like Herkenham,M. 1985. Neuropeptides and their receptors: a psychosomatic mRNAin human leukemia leukocytes and CNS-tissues.Life Sci. 39: network.J. Immunol.135: 820s 107. Blalock, J. E., Smith, E. M. 1985. A 2237 95. Lolait, S. J., Clements,J’. A., Markcompleteregulatory loop betweenthe immuneand neuroendocrine systems. wick, A. J., Cheng,C., McNally,M., Smith,A. I., Funder,J. W.1986.ProFed. Proc. 44:108 opiomelanocortin messenger ribo108. Ahmed,M. S., Llanos-Q, J., Dinanucleic acid and posttranslational prorello, C. A., Blatteis,.C.M.1985.Intercessing of beta endorphin in spleen leukin I reduces opioid binding in macrophages. Y. Clin, Invest. 77:1776 guineapig brain. Peptides6:1149 96. Lolait, S. J., Lim,A. T. W.,Toh,B. H., 109. Boarder, M. R., Erdelyi, E., Barchas, J. D. 1982. Opioidpeptides in human Funder,J. W.1984. Immunoreactive/~endorphinin a subpopulationof mouse plasma:evidencefor multiple forms.J. spleenmacrophages. J. Clin. Invest. 73: Clin. Endocrinol.Metab.54:715 110. Spampinato,S., Goldstein, A. 1983. 277 Immunoreactivedynorphinin rat tis97. Felten, D.L., Felten, S. Y., Carlson,S. sues and plasma. Neuropeptides3:193 U, Olschowka,J. A., Livnat, S. 1985. Noradrenergicand peptidergic inner111. Howlett,T. A., Walker,J., Besser, G. vation of lymphoidtissue. J. lmrnunol. M., Rees, L. H. 1984. "Dynorphin"in plasma:enzymaticartifact and authen135: 755s tic immunoreaetivity.Reg. Peptides8: 98. Smith,E. M., Morrill, A. C., Meyer,W. J. III, Blalock,J. E. 1986.Corticotropin 131 releasing factor inductionof leukocyte- 112. Govoni, S., Salar, G., Colombo, derived immunoreactive ACTHand F., Pasinetti, G., Trabucchi, M.,

Annual Reviews

Annu. Rev. Immunol. 1988.6:219-249. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

OPIOIDS IN THE IMMUNE SYSTEM 249 Mingrino,S. 1986. Episodic secretion of met-enkephalin immunoreactive material in humanplasma and CSF. Pharmacol.Res. Commun. 18:155 113. Janeway, C. A. Jr., Bottomly, K., Horowitz,J., Kaye,J., Jones, B., Tite, J. 1985. Modes of cell:cell communication in the immunesystem. J. Immunol.135: 739s 114. Hazum,E., Chang,K.-J., Cuatrecasas, P. 1979. Specific nonopiatereceptors for fl-eudorphin. Science205:1033 115. Schweigerer,L., Bhakdi, S., Teschemacher,H. 1982, Specific non-opiate binding sites for humanfl-endorphin on the terminal complex of human complement.Nature 296:572 116. Schweigerer,L., Schmidt,W., Teschemacher, H., Wilhelm, S. 1985. flEndorphin:interaction with specific nonopioid binding sites on EL4thymomacells. Neuropeptides6:445 117. Gilman,S. C., Schwartz,J. M., Milner, R. J., Bloom,F. E., Feldman,J. D. 1982. fl-Endorphin enhances lymphocyte proliferativeresponses.Proc.Natl. Acad. Sci. USA79:4226 118. McCain, H. W., Lamster, I. B., Bozzone,J. M., Grbic, J. T. 1982. flEndorphin modulates human immune activity via non-opiatereceptor mechanisms. Life Sci. 31:1619 119. Plotnikoff,N.P., Murgo, A. J., Miller, G.C., Corder,C. N., Faith, R. E. 1985. Enkephalins: immunomodulators. Fed. Proc.44: I 18 120. Murgo,A. J., Plotnikoff, N. P., Faith, R. E. 1985. Effect of methionine-enkephalin plus ZnCI2on active T cell rosettes. Neuropeptides5:367 121. Plotnikoff, N. P., Wybran,J., Nimeh, N. F., Miller, G. C. 1986. Methionine enkephalin: Enhancementof T-cells in patients with Kaposi’s sarcoma (AIDS). Psychopharmacol.Bull. 22: 695 122. Brown,S. L., VanEpps, D. E. 1985. Suppression of T lymphocyte chemotactic factor productionby the opioid peptides fl-endorphin and met-enkephalin. J. Irnmunol.134:3384 123. Brown,S. L., VanEpps, D. E. 1986. Opioid peptides modulate production of interferon ~ by humanmononuclear cells. Cell. Immunol.103:19 124. Simpkins,C. O., Alailima,S. T., Tate, E. A., Johnson,M. 1986. Theeffect of enkephalins and prostaglandins on O~release by neutrophils.J. Sury.Res. 41:645

125. Koff, W.C., Dunegan, M. A. 1985. Modulation of macrophage-mediated tumoricidal activity by neuropeptides and neurohormones.J. Immunol.135: 350 126. Chahl,L. A., Chahl,J. S. 1986.Plasma extravasation induced by dynorphin(1-13) in rat skin. Eur. J. Pharmacol. 124:343 127. Puppo, F., Corsini, G., Mangini, P., Bottaro,L., Barreca,T. 1985.Inttuence of fl-endorphin on phytohemagglutinin-induced lymphocyte proliferation and on the expression of mononuclearcell surface antigens in vitro. Immunopharmacoloyy 10:119 128. Nordlind,K., Mutt, V. 1986. Influence of beta-endorphin,somatostatin, substance P and vasoactiveintestinal peptide on the proliferative response of humanperipheral blood T lymphocytes to mercuricchloride. Int. Arch.Aller#y Appl. Immun.80:326 129. McCain,H. W.,Lamster,I. B., Bilotta, J. 1986. Modulationof humanT-cell suppressor activity by beta endorphin andglycyl-L-glutamine.Int. J. Immunopharmacol.8:443 130. Donahoe,R. M., Madden,J. J., Hollingsworth, F., Shafer, D., Falek, A. 1985. MorphinedepressionofT cell Erosetting:definitionof the process.Fed. Proc. 44:95 131. Carr, D. J. J., Klimpel, G. R. 1986. Enhancement of the generation ofcytotoxic T cells by endogenous opiates. J. Neuroirnmunol.12:75 132. Tubaro, E., Borelli, G., Croce, C., Cavallo, G., Santiangeli, C. 1983. Effect of morphineon resistance to infection. J. Infect. Dis. 148:656 133. Tubaro,E., Avico,U., Santiangeli, C., Zuccaro,P., Cavallo,G., Pacifici, R., Croce,C., Borelli, G. 1985. Morphine and methadone impact on human phagocyticphysiology.Int. J. Immunopharmacol.7:865 134. Wolf,G. T., Peterson, K. A. 1986.Beta endorphin enhances in vitro lymphokineproduction in patients with squamouscarcinoma of the head and neck. Otolaryn#ol.HeadNeckSur#. 94: 224 135. Plotnikoff, N. P., Miller, G. C., Solomon,$. K. T., Faith, R. E., Edwards, L: D., Murgo, A. J. 1986. Methionine enkephalin: Immunomodulator in normal volunteers (in vivo). PsychopharmacoL Bull. 22:1097

Annu. Rev. Immunol. 1988.6:219-249. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Annu. Rev. Immunol. 1988.6:219-249. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Annual Reviews www.annualreviews.org/aronline

Annu. Rev. Immunol. 1988.6:251-281. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Ann.Rev. Immunol.1988. 6: 251-81 Copyright©1988by AnnualReviewsInc. All rights reserved

STRUCTURE AND FUNCTION OF HUMAN AND MURINE RECEPTORS FOR IgG Jay C. Unkeless,

Eileen

Scigliano,

and Victor&

H. Freedman

Department of Biochemistry, Mount Sinai Medical School, New York City, NY 10029 INTRODUCTION The basic mechanisms by which phagocytes recognize foreign organisms have been knownfor more than 80 years. In 1903, Wright & Douglas (1) showed that humanserum contains heat-stabile and heat-labile factors that bind to the bacteria and thus dramatically enhancethe ability of blood phagocytes to ingest Staphylococcus. These factors, IgG and complement, trigger phagocytosis via specific receptors for IgG and complement.Receptors for the Fc portion of IgG (F%R),play a central role in cellular immune defenses and are, in addition, responsible for stimulating the release of mediators of inflammation and hydrolytic enzymesinvolved in the pathogenesis of autoimmunediseases. This review focuses on the current state of knowledgeof the structure and function of the heterogeneous family of the F%Rs of mouse and human. The existence of Fc receptors for IgG (F%R)has been appreciated since the early studies of Berken & Benacerraf (2). However,the recent rapid progress in this field is due in large part to the isolation of monoclonal antibodies (MAbs)specific for different receptors. The Fc~Rfamily probably evolved in parallel with the immunoglobulins;indeed, recent cloning studies (3-5) show that FcrRs form a subgroup of the immunoglobulin supergene family. The FcrRs provide a crucial link between the phagocytic effector cells and the lymphocytes that secrete Ig, since the macrophage/monocyte, polymorphonuclear leukocyte, and NKcell F%Rsconfer an element of specific recognition mediated by IgG. Phagocytic cells thus have several recognition mechanismswhereby friend can be distinguished 251 0732-0582/88/0410-0251 $02.00

Annual Reviews

252

UNKELESS, SCIGLIANO & FREEDMAN

from foe, including the FcrRs, the complement receptors, and receptors for oligosaccharides.Sincethere havebeenseveral reviewson F%Rs (6, 7), wepresent primarily recent data on FcrRreceptor structure and function.

Annu. Rev. Immunol. 1988.6:251-281. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

NOMENCLATURE A nomenclaturefor the family of Fc receptors was recently discussed at the meeting on Fc receptors and ImmunoglobulinBinding Factors sponsored by FASEB in June, 1987. A nomenclaturesubcommitteeagreed uponthe followingdefinition for Fc receptors: "A group of surface membrane molecules that specifically recognize and bind homologousimmunoglobulin via the Fc portion, and which putatively mediate biologic functions." The nomenclature we have adopted for this review--summarizedin Table 1--is consistent with that provisionally agreed to by those present. For convenience,other namesby whichspecific receptors havebeendesignatedare also listed. Thespecies of origin is designatedby twolowercase letters precedingthe receptor (e.g. mofor mouse,rt for rat, ra for rabbit, hu for human)and the principal immunoglobulin isotype, the bindingof whichhelps to characterize the receptor, is indicated by a subscript Greekletter. TheRoman numeralsrefer to distinct isotypes of the receptor. Until genetic (sequence)data becomeavailable, this aspect of the nomenclature mustbe consideredtentative. This is particularly true with respect to cross-speciesrelationships.

HUMANFc RECEPTORS huFcrRI Human leukocyteshaveat least three different receptors for IgG. huFc~RI, the receptor found on monocytesand macrophages,binds monomericIgG with high avidity, as reported by Huber & Fudenberg(8) and by Huber et al (9). Therank order of binding is IgG1> IgG3> IgG4>> IgG2, with a Kaof --~ 1-3 x 10~. Competitivebinding studies performedby Anderson & Abraham (10) of different subclasses of IgGto the U937cell line show oneclass of high avidity bindingsites. Theprotein responsiblefor the high avidity bindingis a 72,000M r peptide isolated by affinity chromatography on IgG-Sepharose(Anderson,11). Thesameprotein was later isolated immunoprecipitation with a goat antiserum obtained following immunization with afffinity-purified Fc-bindingproteins (Anderson et al, 12). r~ONOCLONAL ANarmODIES Recently, Andersonet al (13) have isolated monoclonalantibody (MAb32.2)that immunoprecipitatesthe 72 kd protein from U937cells. However,MAb32.2does not inhibit IgG binding,

Annu. Rev. Immunol. 1988.6:251-281. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Annual Reviews FC~ RECEPTORS 253

Annual Reviews

Annu. Rev. Immunol. 1988.6:251-281. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

254

UNKELESS, SCIGLIANO & FREEDMAN

indicating that it is directed against an epitope distant from the receptor’s ligand binding site. Similarly, the production of superoxide by monocytes . initiated by immunecomplexes was not blocked by MAb32.2. Frey & Engelhardt (14) report a new MAb, FR51, that immunoprecipitates the same 70,000 Mr protein found with MAb32.2. However, unlike MAb32.2, FR51also inhibits binding ofmonomericand aggregated IgG to U937 and HL-60 cells but has no effect on binding of IgG to granulocytes or Raji cells, which do not bear huF%RI.The F(ab’)2 fragment of FR51also inhibited immunecomplex binding, although a higher concentration of antibody was needed. These investigators found that huF%RIis strikingly trypsin resistant, and that endoglycosidase F treatment to remove N-linked carbohydrate resulted in a core protein of 40,000

Mr.

VAeENCV According to O’Grady et al (15), the valence ofhuF%RIis one. They saturated U937cells with mixtures of labeled and unlabeled IgG1 x and 2, lysed the cells, and adsorbed the lysates on an antikappa immunoadsorbent. They reasoned that if the huFcyRI was multivalent, some labeled lambda IgG1 wouldbe precipitated by the antikappa adsorbent, but essentially no labeled IgG1 lambda was found. However, in the presence of MAb32.2, which would render the receptor bivalent (since the mAb is bivalent), binding of labeled IgG1 lambda to the antikappa immunoadsorbent was observed. OF huFcrRI huF%RI is likely to be centrally involved in antibody-dependent cellular cytotoxicity (ADCC).Guyre et (17) have shown huFc~RI on peripheral blood monocytes and the U937 cell line is induced 8-10 fold in 8-12 hr by y-interferon (IFN-~). This striking result suggests that huFcrRI mayplay a role in enhancedeffector function often associated with IFN-7 stimulation [See Schreiber (16) for review]. In support of this, Akiyamaet al (18) demonstratethat ~-interferon treatment of U937 cells induces mouse IgG2a- and IgG3-dependent ADCC. Additional compelling evidence for the role of huF%RIin ADCCis provided by Shen et al (10). They report that covalent heteroconjugates formed with intact 32.2 MAbor its Fab fragment, and antibody directed against chicken erythrocytes (E), mediate efficient ADCC by monocytes and U937 cells. This ADCCwas not due simply to formation of cellcell conjugates, since heteroconjugates formed from anti-HLA and antichicken E IgG did not trigger ADCC.The extent of the ADCCwas enhanced by pretreatment of the cells with IFN-y. Significantly, the ADCC mediated by the heteroantibody was not inhibited by IgG at 2 mg/ml, in striking contrast to the inhibition of anti-chicken E IgG-mediated ADCC EFFECTOR FUNCTIONS

Annual Reviews

Annu. Rev. Immunol. 1988.6:251-281. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

FCr RECEPTORS 255 by 40 ~ug/ml of IgG. Similar results were reported by Graziano & Fanger (20), who found that monocyte ADCCagainst hybridoma cells bearing MAb32.2 on their surface was stimulated by IFN-y, but no ADCCwas seen against hybridomas bearing MAbsdirected against other antigens such as the iC3b receptor or class I histocompatibility antigens. IFN-y also induces huFcrRI on neutrophils, which normally do not express this receptor. Following induction with IFN-~, neutrophils will mediate ADCC triggered by MAb32.2 Fab/anti-chicken E Fab heteroconjugates (20a). The results provide a possible rationale for specific anti-tumor/antihuFcyRI heteroconjugates in immunological therapy of malignancy, assuming that appropriate antibodies directed against tumor antigens are selected to form the heteroconjugates. Undoubtedly,other agents besides IFN-~affect the level and/or activity of Fc~Ron cells. For example, Girard et al (21) have shown that dexamethasone, which by itself decreases huFcrRI expression by monocytes, further elevates huFcrRI levels induced by treatment of monocytes with IFN-~. Yanceyet al (22) report that CSa, an important cytokine released upon activation of complement, potentiates the ability of monocytes to bind IgG-sensitized and C3b-sensitized sheep E. The activation by CSa was rapid (30 min), suggesting that receptor synthesis is not required. Another chemotactic peptide, F-met-leu-phe, had no effect on rosetting of E sensitized with either C3b or IgG. huFcrRII A second receptor on humanleukocytes recently described is huFc~RII, which was initially identified by affinity chromatographyof U937lysates on IgG-Sepharose (11) and then immunoprecipitated with a goat-antiFcrRantibody elicited by immunizationwith affinity purified protein (12). MAbIV.3, directed against huFcrRII, was isolated by Rosenfeld et al (23). MAbIV.3 immunoprecipitates a protein of 40,000 Mr. The antigen is distributed on a wide variety of cell types, including monocytes,platelets, neutrophils, possibly B cells, and the K562cell line. The only huFcrR receptor detected on K562cells and on platelets is huFcrRII. The IV.3 MAb(and the Fab fragment) block aggregation of platelets induced IgG aggregates but have no effect on aggregation triggered by other agents such as thrombin or collagen. Kulczycki (24) finds that neutrophils and eosinophils have different FcrRs and reports that the Mr of the FcrR on eosinophils isolated by IgG affinity chromatography is 43,000. This molecule is probably huFc~RIIsince Looney et al (25) report that this receptor is present on eosinophils. The huFcrRII has low avidity and is the humanreceptor responsible for the binding of murine IgG2b aggregates. Jones et al (26) report that

Annual Reviews

Annu. Rev. Immunol. 1988.6:251-281. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

256

UNKELESS,SCIGLIANO& FREEDMAN

binding of this ligand to U937cells was greatly enhancedby lowering the ionic strength of the medium.In contrast, the binding of murine IgG2ato U937cells was unaffected by changes in ionic strength. The binding of the IgG2b aggregates was poorly inhibited by murine IgG2a and vice versa. All of these properties serve to distinguish the low avidity huFc~RIIfrom the high avidity Fc~RI, although both receptors are present on U937cells. McCoolet al (27) examined binding of IgG to K562 and U937 cells and found K562 had too low an affinity for IgG to measure the binding of monomeraccurately. High avidity binding of murine IgG2a, but not IgG2b, to U937 cells was found. Studies with switch mutants of mouse myelomas demonstrated that binding of IgG2a to human Fc~R required, in addition to the CH3domain, amino-terminal domains of mouse IgG. huFc~RII POLYMORPHISM The role of huFc~RII, found on an extraordinary variety of cells, is not well understood, but somerecent experimentsof Andersonet al (28) suggest that it is involvedin T cell proliferative responses driven by murine IgG1 anti-Leu-4 (T3) MAbs.Tax et al (29) reported that in 30%of otherwise normal individuals IgG1 anti-T3 MAb failed to simulate mitosis of T cells in the presence of monocytesalthough these antibodies were mitogenic when coupled to Sepharose. Monocytes from nonresponding individuals did not rosette with IgGl-coated E (Tax et al, 30), although they do express huFcrRII. Similar results suggesting the presence of multiple receptors on monocyteswere published by Clement et al (31), who found that unresponsiveness to IgG1 anti-T3 antibodies was not absolute but could be overcome by addition of high concentrations of antibody. The monocyte-dependent mitogenic response is thought to be Fc dependent and probably occurs through cross-linking of the antigen on the T cell surface. The polymorphism in response to IgG1 anti-T3 discussed above is likely to be mediated by the huFcrRII, since this response is blocked by an anti-huFcrRII MAb(Looney et al, 32). Supporting this role of huFcrRII, Andersonet al (28) find a polymorphismin the isoelectric point of huFc~RIIthat correlates well with the observed gene frequencies for IgG1 anti-T3 nonresponsiveness in the population (29-31). A different type of polymorphismis reported by Rosenfeld et al (33), who find that reproducible differences in the amount of huFc~RII on platelets from different individuals that correlate with the sensitivity of platelet aggregation by IgG aggregates. The huFcrRII mayserve as a target for NKcell activity, an interesting hypothesis in light of the widespread distribution of this antigen. Perl et al (34) report a correlation between huFc~RII density, determined binding of anti-FcrRII, and susceptibility to NKcells. Additional direct

Annual Reviews

Annu. Rev. Immunol. 1988.6:251-281. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

FC~ RECEPTORS

257

evidence for huF%RIIas an NKtarget is the observation that intact IV.3 MAband its Fab fragment partially block NKactivity directed against K562and U937 cells. The transferrin receptor is also thought to be a recognition site for NKcytolysis (Vodinelich et al, 35; Alarcon &Fresno 36). Perl et al (34) find that a combination of anti-transferrin receptor MAband anti-huF%RII MAbcan totally block NKcytolysis of K562 cells. As would be predicted, the anti-huF%RII MAbhas no effect on cells not bearing the epitope. Single cell binding and cytolysis experiments suggest that the transferrin receptor and huF%RIIserve as recognition elements for K/NKcells and do not play a role in subsequent lyric events. huFcrRIlI A third Fc~Ridentified on humanleukocytes is a low avidity F%Rwith very broad electrophoretic mobility from 50,000-70,000 M~in SDS-acrylamide gels (huF%RllI). This receptor, huF%RIII, has recently been assigned CD16(37) and is present on a variety of different cell types, including neutrophils, NKcells, and tissue macrophages (38--42). The receptor appears late in the differentiation of granulocytes and monocytes.Fleit et al (43) found that huF%RIII was present in bone marrow cells at the metamyelocytestage, but not earlier. In agreement with this observation, F%RIII was not detectable on uninduced HL-60cells, which are arrested at the promyelocyte stage, but it was induced by retinoic acid and dimethylsulfoxide, both of which drive HL-60toward a more mature neutrophil morphology. Similarly, monocytes do not express huFcvRIII, but the antigen is expressed at high levels on tissue macrophagespresent in liver, spleen, and lung. Culture of blood monocytesin vitro for one week, which results in cells that have manymacrophage-likecharacteristics, results in induction of the antigen. No agents were found that induce huF%RIIIon U937, and the factor(s) that induce the antigen on tissue macrophages vivo are not known. MONOCLONAL ANTIBODIES There are several MAbsdirected against the CD16, or huF%RIIImolecule, including 3G8, B73.1, Leu-11 a,b, and VEP13 (Perussia et al, 41). These MAbsare directed against different epitopes on the receptor and thus differ in the efficacy with which they block binding of IgG complexes. MAb3G8 is the most efficient inhibitor of antibody binding but is unsuitable for ablation of populations of cells by complement cytotoxicity, since it is a mouseIgG1 and fixes complementpoorly. Leu-I lb, a mouseIgM, is preferable in this regard. B73.1 was initially isolated as a reagent specific for NKcells and was reported to bind to neutrophils of 50%of individuals, suggesting that the MAbrecognizes an alloantigen. However, B73.1 reacts with all NKcells regardless of its

Annual Reviews

Annu. Rev. Immunol. 1988.6:251-281. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

258

UNKELESS, SCIGLIANO & FREEDMAN

reactivity with neutrophils from the same individual. Werneret al (37) have found that NA1 and NA2antigens, reported to be neutrophil-specific, are huFc~RIII alloantigens. A good correlation has been found between autoimmune neutropcnia and NA1/NA2-specific antibody in patients’ sera--see Madyasthaet al (44), and Lalezari et al (45) for review. There is, however, no information on the presence ofNA1 and NA2determinants on tissue macrophages. Tetteroo et al (46) have examined a panel of CD16antibodies for react~ivity with different cell types and found they can be divided into five classes: (a) NA~-specific MAbsthat do not react with NK/Kcells; (b) MAbsthat bind to both NA~and NA2 PMNbut not to NK/K cells; (c, d) two groups that stain, albeit with different fluorescence intensity, both NA~and NA2on PMNand NK/Kcells; (e) a group of antibodies that stain NK/Kcells much more brightly than the NA1and NA2PMN. The biochemical differences responsible for the patterns of staining are not yet understood, but when structural data are available it may be possible to distinguish between the huFc~RIII present on the NK/Kcell, the neutrophil, and the macrophage. Clarkson & Ory (47) suggest that the huFc~RIII present on neutrophils is different from that found on tissue macrophages. Immunoprecipitation with MAb3G8 of 125I labeled protein from neutrophils resulted in a protein having a broader electrophoretic mobility on SDSoPAGE than the huFc~RIII isolated from monocytes cultured in vitro for three weeks. Moreover, following digestion with N-glycanase, the 50,000-70,000 Mr neutrophil protein was cleaved to a 33,000 Mrspecies, whereas the macrophage 55,000 Mr protein was not altered in mobility. This striking difference suggests that either the protein core or the glycosylation of the receptor is quite different for huFc~RIIIisolated from the two cell types. huFc~RIII is a receptor of low avidity that preferentially binds immune complexes, but not monomericIgG. Kurlander & Batker (48) found that neutrophils, which do not express the high avidity huFc~RI, bind IgG1 dim¢rs with an avidity 100-1000-fold lower than do monocyt¢s. These results were confirmedby Fleit et al (38), whofound negligible binding monomericIgG1 to neutrophils, although these cells obviously bear Fc~R and saturate with MAb3G8 at > 1 x 105 sites per cell. Kurlander et al (49) have shown, by Scatchard analysis of the binding of IgG1 dimers monocytes and peritoneal macrophag~s, that the macrophages express a new low avidity IgG binding site not present on blood monocytes. IgG1 binding to humanperitoneal macrophagesyields biphasic Scatchard plots with a low avidity site (1.1 x 107 M-l) and a high avidity site with a Ka 200-fold higher. The number of low avidity sites on the peritoneal macrophageswas > 2 x l05 sites/cell, some five-fold higher than the num-

Annual Reviews

Annu. Rev. Immunol. 1988.6:251-281. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

FCv RECEPTORS

259

ber of high avidity sites presenton the samecells. Clarksonet al (50) found strong immunoperoxidase staining with MAb3G8in sites wherethere are high concentrations of tissue macrophages--inthe red pulp of spleen and in Kupffercells of the liver. FUNeTIO~q OFh’uFcvRIII F%R’sclearly play a role in the clearance of immune complexes.Oneapproachto dissection of the role of the various humanF%Rspecies is to study the effect on immunecomplexclearance of in vivo administration of MAbsthat inhibit FcvRfunction. This approach has been used by Clarkson et al (50) to study the role huF%RIIIin vivo. The epitope recognized by MAb3G8is present only on humanand chimpanzeeleukocytes, necessitating the use of chimpanzees for the in vivo experiments. The clearance of [51Cr]O4-1abeled c serumwas measured autologous E sensitized with chimpanzeeanti-aCbCd in lightly anesthetized animals, followingthe methodology developedby Frank et al (51) for performingclearance studies in humans.Infusion MAb 3G8at doses as low as 0.25 mg/kgbodyweightresulted in a dramatic (20-fold) increase in clearance time of the sensitized E comparedto the pre-infusion clearance T1/2 of 1 hr. Infusion of 3G8IgGresulted in a transient (4-day) neutropenia, which mayhave been due to the opsonization and clearance of the neutrophils by intact 3G8IgG. Infusion of the Fab fragment of 3G8also blocked clearance but did not result in neutropenia, although the density of 3G8Fab on peripheral neutrophils was similar to that found with the intact antibody. These experiments demonstratethat the FeaRprimarily responsible for clearance of the IgGsensitized E is huFc~RIII,althoughthey do not rule out a lesser role for other F%Rand complementreceptors. ADCC of K/NKcells is also thought to be mediated by huF%RIII. Several groups have examinedthe potential of different subclasses of murine IgG to mediate ADCC of humanlarge granular lymphocytes (LGL).Anasetti et al (52) isolated a series of anti-Thy-1.1switchvariants and found that the rank order of activity in mediatingADCC by LGLwas IgG3 > IgG2a> IgG2b, with murine IgG1essentially inactive. Similar results werereported by Kippset al (53), whoused switchvariants. Clearly other factors determineeffectiveness for a given antibody in mediating ADCC reactions, however. Christiaansen at al (54) covalently coupled humanf12 microglobulin to murine lymphocytesand comparedthe sensitivity of these cells, andthe human cell line 8866(whichexpresseshuman f12 microglobulin), to humanK cell-mediated ADCC mediated by an IgG2aantihumanf12 microglobulin. Althoughthe humanand murinecells boundthe same amountof the sensitizing anti-human//2 microglobulin MAb,humanK cells did not lyse the mouselymphocytesregardless of the antibodyconcentration,whereasthe 8866line wasvery sensitive to lysis.

Annual Reviews

Annu. Rev. Immunol. 1988.6:251-281. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

260

UNKELESS, SCIGLIANO & FREEDMAN

Titus et al (55) examinedK/NKcell ADCC mediated by heterocrosslinked anti-huFevRIII MAb3G8 and either anti-DNP or anti-tumor MAbs.The anti-huF%RIII cross-linked to anti-tumor MAbwas capable of targeting Kcells to kill tumorcells andwaseffective in preventingtumor growth of melanomacells in a nude mousein a Winn-typeassay. Tumor cell cytotoxicity requires cross-linking via F%RIII.Heteroconjugates containing anti-HLAclass I antibody were not effective. Shenet al (20a) also find that neutrophil ADCC can be triggered by heteroconjugates containing anti-huFcrRIII Fab fragments. It appears that neutrophil and K/NKADCC activity can be directed effectively by heteroconjugatescontaining an anti-huFcrRIII MAb,a system analogous to the monocyte ADCC system, whichis triggered by anti-huF%RIheteroconjugates, and the CTLsystem, triggered by anti-T3 heteroconjugates. The phagocyticpotential of huFcrRIIIon neutrophils can be modulated by a small (< 10,000M~)cytokine whichwasfirst noted as a stimulator of the C3breceptor for phagocytosis. This low molecular weight lymphokineis released from T cells in response to humanmonocytesincubated with immunecomplexesand is released constitutively from humanT cell line MO(t)(56, 57). Gresham et al (58) report that the cytokine rapidly stimulates phagocytosisbut does not elevate the binding of IgG-sensitized E by PMN(Greshamet al, 58). Binding of the anti-huF%RIII MAb3G8 to neutrophils wasalso unaffected by the cytokine. However,Greshamet al (58) describe a MAb against neutrophils that inhibits only the portion of the phagocyticindex stimulated by the cytokine. Thebinding of immune complexeswas totally inhibited by MAb 3G8. Theseresults suggest that there are functionally different classes of huF%RIII on the surface of the neutrophil. Jack & Fearon (59) find that 50%of PMN huF%RIIIco-caps with C3b receptor, implyingthat FcrRmayexist in different states dependingon whetheror not it is associated with the cell cytoskeleton. There is no evidence for phosphorylation of F%Rs,although if this is a transitory event, it mighthave beenmissed. Altered Expression of Receptor and Autoimmune Disorders In addition to the clearly defined polymorphisms of the huFcrRIImolecule, and the neutrophil huFc~RIIIalloantigens discussed previously (28, 29, 33, 37, 46, 47), there are studies documenting variability in receptoractivity linked to histocompatibilityantigens. Lawleyet al (60) report an association between a depressed rate of IgG-complexclearance and the HLAB8/DRw3 haplotype, which the haplotype found in 90%of patients with dermatitis herpetiformis.In addition Salmon et al (61) report statistically significant decreased F%R-mediated phagocytosisby monocytesfrom nor-

Annual Reviews

Annu. Rev. Immunol. 1988.6:251-281. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

FC~, RECEPTORS

261

real individuals bearing DR2and DR3or DQwlantigens. Salmon & Kimberly (62) also find that monocytesfrom these individuals have decreased ability to phagocytize concanavalinA-coatedrabbit E. They suggest that the receptors bear carbohydrate that interacts with concanavalinA, resulting in a phagocyticstimulus. In accordwith the hypothesis, they find a dramaticinhibition in phagoeytosisof both IgG-sensitized E and concanavalinA-sensitized E following adherence of monocyteson aggregated IgG coated coverslips. Underthese conditions, there is no inhibition of phagocytosisof E treated with tannic acid or wheatgerm lectin, nor is phagocytosisof zymosan affected. In several diseases associated with immunecomplexessuch as systemic lupus erythematosus(SLE)(51), Sjogren’s syndrome(63), and dermatitis herpetiformis(60), there is a significant depressionof clearance of IgGsensitized erythrocytes.Theseverity of the disease in SLEcorrelates with depression of immunecomplexclearance and remission of the disease is accompaniedby a return of more normal clearance rates. In Sjogren’s syndrome,prolongedclearance time of IgG-sensitized erythrocytes correlates with diffuse tissue damage,whereaspatients with normalclearance times have disease limited to exocrineglands alone. Thechangesin FeaR activity on circulating monocytes in SLEpatients are likely to be complex. Salmonet al (64) find that the numberof IgG-sensitized erythrocytes boundto monocytesfrom SLEpatients actually increases, while the number ingested decreases. It is possible, however,that these assays, which predominantlymeasurehuFc~RIactivity on monocytes,are not the most germane,since most of the clearance of immune complexesoccurs in the spleen and liver by macrophages,whichbear large amountsof huFc~RIII. Immunethrombocytopenic purpura (ITP) is an autoimmunedisease associatedwith the presenceof elevated levels of platelet-associated immunoglobulin.This results in increased clearanceof opsonizedplatelets from the circulation, with subsequentthrombocytopenia and bleeding. Clinical managementof the disease includes splenectomy, which removes the primarysite of clearance, and a site of antibody production, corticosteroids, whichmayact in various waysto dampenmononuclearphagocyte function, intravenous high dose gamma globulin (Bussel, 65), and most recently danazol, a limited androgen(Schreiber et al, 66). Intravenous gamma globulin is reported by Salmonet al (67) to decrease phagocytosis of IgG-sensitized erythroeytes by monoeytes,and danazolis reported to decrease the numberof huFc~RIsites on monocytes. The ability of the anti-huFc~RIII MAb3G8to block immunecomplex clearancein the chimpanzee led Clarksonet al (68) to test the therapeutic usefulness of this MAb in patients with ITP. In the one reported case, infusion of 3G8at 1 mg/kgin a patient with ITP refractory to all therapy

Annual Reviews

262

UNKELESS, SCIGLIANO & FREEDMAN

Annu. Rev. Immunol. 1988.6:251-281. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

led to a dramatic rise in platelet count to normallevels for 2 weeks.A secondinfusion of MAb 3G8led to a smaller but still clinically significant rise in platelet count. Prolongedclearance of anti-Rh sensitized erythrocytes was demonstratedfollowing infusion of MAb3G8in this patient. Althoughthe dramatic increases in platelet numberdid not persist, the patient did stabilize with a somewhat higher platelet count than prior to 3G8infusion, and the patient onceagain becameresponsiveto intravenous gamma globulin. Thetherapeutic efficacy of 3G8infusions for ITP is still underinvestigation. Complement and Fc~R Interactions Many factors affect the ability of cells to phagocytizeopsonizedparticles. Already mentioned were the effect of IFN-y on receptor number(17) and lymphokineson phagocytosis (58). Ehlenberger& Nussenzweig(69) demonstrateda synergistic effect of complement and IgGon phagocytosis by macrophages.It is nowclear that membrane Clq and Fc~Rsact synergistically to promotephagocytosis and ADCC. Loos(70) and Heinz et (71) demonstratedthat Clq on macrophagesmayfunction as an Fc~R, showing inhibition of the binding of IgG coated E by intact and F(ab’)2 polyvalent and monoclonalanti-Clq antibodies. Theseresults are echoedin recent workby Bobaket al (72), whofind that Clq coated surfaces markedly enhance the phagocytosis of IgG-opsonized targets by monocytes.The stimulation is blocked by anti-Clq F(ab’)2 and is not seen in polymorphonuclearleukocytes. Mocharlaet al (73) report that inhibitors of collagen biosynthesis (whichalso inhibit synthesis of Clq) block Fc~Rfunction but have no effect on C3breceptor function. That these interactions are significant is suggested by results of Hamada& Greene(74) whofind that Clq enhancesIgG-dependentkilling of Schistosomamansoni.The collagenousdomainof C l q is thought to be responsible for the enhancedkilling and phagocytsosisseen in the two systems. Fc~R-Mediated "’Sideways" ADCC ÷ Several groups have observed that while anti-CD3 MAbsbound to CD3 cytotoxicT cells inhibit antigen-specificCTL-mediated cytolysis the antiCD3MAbswill also promote the killing of Fc~R-positive target cells (Mentzerat al, 75; Spits et al, 76). Interestingly, only T3antigen-specific MAbs wereeffective in triggering this target cell Fc~R-dependent cytolysis. A similar phenomenon was observedby Leeuwenberg et al (77) and Egawa et al (78). Anti-huFc~RIII MAbsof appropriate subclass bound huFc~RIIIpositive effector cells (either T3 positive or negative)will kill target cells bearing Fc~Rs.Daudicells, whichhave only a low avidity Fc~Rspecific for murineIgG1, were not killed by NKcells armedwith a

Annual Reviews F% RECEPTORS

263

Annu. Rev. Immunol. 1988.6:251-281. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

murineIgG2aanti-huFcrRIII MAb,but they were killed by similar cells coated with a murine IgG1 anti-huF%RMAb.In contrast, U937cells, whichexpress both huF%RIand huF%RIII,were killed by NKcells armed with both IgG1 and IgG2a anti-huF%RIII MAbs. MURINE Fcr RECEPTORS moFc~Rheterogeneity In the mouse, as in humans,multiple F%Rshave been identified, but manyquestions regarding their function and structure remainunanswered. Previous work, reviewedby Dickler (7) and by Unkelesset al (6), led the identification of three receptors on mouseleukocytes, moF%RI binds murine IgG2a and humanIgG1 with high avidity; it is found only on macrophages(79) and is somewhattrypsin sensitive (80). Diamond Scharff (81) and Unkeless (82) demonstrateda second F%Rspecific mouseIgG2b and IgG1 (moF%RIII),and Diamond& Yelton (83) characterized an F%Rspecific for IgG3 (moF%RIII).The identification these receptors wasaccomplishedlargely by competition experimentsin whichthe binding of ligands (usually erythrocytes coated with monoclonal anti-RBCantibody)wasinhibited by addition of aggregatedIg of different subclasses, andby inhibition studies using proteolytic enzymesor an antiF%RMAb2.4G2 .(Unkeless, 82). The assignment of specificity for the F%Rrecognized by 2.4G2 as IgG2b/IgG1maybe too narrow. It is clear from several reports (84-87) that MAb2.4G2 recognizes, in addition to a low affinity receptor on macrophages,an analogous receptor on lymphocytes.In those studies, however, the lymphocytereceptor reactive with MAb2.4G2 boundIgG1, IgG2b, and IgG2a. The lymphocyte/macrophage F%Rhas now been cloned (see below)and expressedin non-FeaRbeating cells. Theresults unequivocally demonstrate (R. Weinshank,J. Ravetch, J. C. Unkeless, unpublishedresults) that the receptor that binds MAb 2.4G2also binds IgG2a with low avidity. The moF%RI on macrophagesprobably masked the weaker IgG2abinding activity of moFcrRII,but this low avidity binding was detected both on melanoma cells transfected with moFc~RII and on lymphocytes,whichlack the high avidity moF%RI. The evidence from cloning studies to date suggests that low avidity macrophageand lymphocytereceptors reactive with MAb2.4G2 are, in their extracellular domains,identical in aminoacid sequence.However, recent studies by Kulczyckiet al (88) suggest that the Fc~Ron mouse suppressor T cell hybridomas,whichreact with the anti-F%RMAb2.4G2, differ in specificity fromthoseon B cells, since the receptorfromthe T cell hybridomasdoes not bind either IgG3 or IgG2a. The size of the T cell

Annual Reviews

Annu. Rev. Immunol. 1988.6:251-281. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

264

UNKELESS, SCIGLIANO & FREEDMAN

receptor from125I-labeledcells was56,000-61,000Mr,whichis consistent with previousreports of the size of the F%R isolated fromthe $49.1T cell line by Mellman&Unkeless(89). Thebiochemicalbasis for these differencesin specificity is unclearbut is likely to be dueto differencesin posttranslational modificationrather than to different genes. Progress on the characterization of the moF%RI and moFcrRIIIhas been slow. Lane& Cooper(90) isolated, by affinity chromatography,Fcr binding proteins from IgG2a, IgG1, and IgG2bimmunoadsorbents;they found the IgG2a-bindingproteins were somewhatmore acidic and also slightly different in size fromthe proteins that boundto IgG2band IgG1. Nothinghas beenreported on the characterization of the proteins responsible for IgG3binding. Oneof the moreintriguing recent results is the demonstrationby two independentgroups(Holmeset al, 91; Hibbset al, 92) that the Ly-17locus defines a polymorphismof the FcrR recognized by MAb2.4G2. Ly-17 antisera block Fc~Ractivity and whenused in combination with MAb 2.4G2in preclearing experiments,showclearly that these sera recognize different determinantson the samemolecules.This is of interest because Ly-17(called Ly-m20.2 earlier by Market al, 93) is tightly linked to Mls. Disparities at Mls trigger a strong mixedlymphocytereaction mitogenic responsebut do not generate cytotoxic T cells. Ly-17mapsto the distal arm of chromosome I, as does the gene for cloned murine F%R(Ravetch et al, 3). Theseresults wouldseemto be contradictory to results of Baum et al (87), whofound that a polymorphismof murinc B cell F%Rthat results in an altered affinity for rat IgGmapped to chromosome 12 distal to the Igh locus. Perhapssecondarybindingsites such as the Clq interaction discussedearlier accountfor these results. Domains Involved in IgG Binding To Fc~R The CH2domain of IgG is generally thought to be the domain that interacts with moF%RI, and it seemslikely that carbohydrate plays an important role in maintenanceof proper conformationof IgG for binding to FcrR. Nose& Wigzell(94) report that aglycosylated IgG2a, isolated by growingmyeloma cells in the presence of tunicamycin, was unable to fix complementor to bind to macrophageF%R.Leatherbarrowet al (95) also find that oligosaccharidesplay a role in maintenance of conformation required for binding, since aglycosylatedIgG2aboundwith a 50-fold lower affinity to humanmonocytes.However,the capacity of the aglycosylated IgG2ato activate C1 was approximatelythe same as that of the native protein. Diamond et al (96) haveused a series of switch variants to examinethe domains that interact with the different F%Ron mousemacrophages.

Annual Reviews

Annu. Rev. Immunol. 1988.6:251-281. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

FCr RECEPTORS

265

They conclude that IgG2b binds via the Ca2 domain, since a mutant IgG2b with a deleted CH3still bound to the moFcrRII. Another mutant with an IgG2b-like Cry2 domain and an IgG2a-like CH3 domain bound to both moFcrRIand moFcrRII. The situation is clearly complex, however, because in a later paper Birshstein et al (97) describe a mutant with IgG2b-like CH1 and hinge region, and IgG2a-like C~2 and CH3region, which does not bind to moF%RI,although the Fc fragment of the mutant IgG did. The level of discrimination of the moFcrRIand the moFcrRIIis remarkable. Diamondet al (98) looked at the inhibition of rosetting EIgG2a and EIgG2b to macrophages by cyanogen bromide fragments of IgG2a and IgG2b. Specific inhibition was found for IgG2a and IgG2b by homologous CNBrfragments from the CH2 domain of the antibodies. The inhibitory CNBrpeptides from IgG2a and IgG2b differed by only four residues in 62 amino acids. Only one peptide from IgG2b was inhibitory, but two IgG2a peptides, one from the C~2 domain and one from the CH3 domain were inhibitory for EIgG2abinding. These results suggest that there maybe two domainsthat are involved in separate binding sites for the moFcvRI. Binding of both IgG2a CNBrpeptides was sensitive to trypsin, as was predictable from previous studies (79-81). Function

of Murine FcrR

In contrast to someother receptor-ligand systems, such as the transferrin receptor and the asialoglycoprotein receptor, the murine FcrR reactive with MAb2.4G2 apparently is not recycled following internalization of immuneaggregates. Mellman et al (99) found that as much as 50% macrophageFcrR could be driven inside the cell by presentation of IgGsensitized erythrocyte ghosts,, and the amountof FcrR on the cell surface remained depressed for many hours, whereas a series of other plasma membraneantigens was unaffected. Furthermore, the T1/2 of the ~25Ilabeled FeaR internalized along with immunecomplexes was 2 hr, compared to a TI/2 of 10 hr in the absence of ligand. Evidence from several groups has suggested that FcrR bound with monomeric ligand (i.e. IgG) does not deliver ligand to the endosomal compartmentbut rather recycles to the cell surface with bound antibody (Kurland & Gartrell, 100; Jones et al, 101). The determining factor whether bound ligand is cleared to the lysosomes seems to be the valence of the complex. Monomersand dimers were poorly internalized whereas larger complexeswere efficiently catabolized. Ukkonenet al (102), Mellman et al (103), and Mellman& Plutner (104) demonstrate that monomeric 2.4G2 Fab fragment is not taken into lysosomes and degraded, but cycles with the receptor through the endocytic compartmentand is found in low density endosomes. Whenthe 2.4G2 Fab was rendered multivalent by

Annual Reviews

Annu. Rev. Immunol. 1988.6:251-281. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

266

UNKELESS, SCIGLIANO & FREEDMAN

adsorption to colloidal gold, it was transferred to the lysosomalcompartment, indicating once again the importanceof valence in lysosome clearance. The Fc~R recognized by MAb2.4G2, like the huF%RIII described previously, is a low avidity receptor whichbinds monomeric IgGpoorly. Studies by Kurlanderet al (105) and Kurlander& Hall (106) have established that this FcrR is involved in clearance of immunecomplexes, since infusion of MAb2.4G2 in mice was a potent inhibitor of F%R function. It is, however,difficult to assign functionto the different receptors, since all of themoften coexist on the samemacrophage,and since the isotype specificity as discussedpreviouslyis relative, not absolute. Ralphet al (107) found that all classes of murineIgG mediatephagocytosis andlysis of erythrocytes, but to extrapolate fromthat experimental result to the conclusion that all FcvRsmediatephagocytosismaynot be warranted. However,someevidencesuggests that subclass specificity is important in ADCC mediated by thioglycollate-elicited macrophages.Results of Matthewset al (108) and of Herlyn & Koprowski(109) both implicate IgG2aas the subclass of IgG that mediates marcophageADCC. In these studies, both groups used either an in vivo nude mousemodelor a homologousanimal tumor model to demonstrate protection by anti-tumor IgG2a. Langlois et al (110) have also developedan in vitro modelthat mimicsthe in vivo results on the efficacy of IgG2aantibody. In addition, Nathan et al (111) found that MAb2.4G2 inhibits ADCC mediated BCGstimulated macrophages70%. Thus, both the low avidity macrophage moF%RII and the high avidity moF%RI are implicated in ADCC. Thefunctionof Fc~Ron B cells is still not clear. Thestudies of Phillips & Parker (112-t 14) suggest that formation of immunecomplexesin vivo are inhibitory for B cell differentiation, as wouldanti-idiotypic antibodies reactive with surface Ig, if they bound to F%R.They and others had observed that rabbit anti-IgG would not trigger a mitogenic B cell response, although the F(ab’)2 fragmentwas stimulatory. However,if the F%R was occupiedby MAb2.4G2, then the intact rabbit anti-/~ antibody wasstimulatory. Thus, they. concludethat for anti-# antibodyto inhibit effectively a mitogenicB cell response, it has to forma ternary complex with the surface Ig and F%R.Howthe formation of this ternary complex differs frombinding of the anti-FcrR MAb with respect to FcrRsignalling is unclear. It has been suggested that FcrR producesa signal by forming an ion channel, thereby depolarizing the macrophage.Younget al (115) used tetraphenyl phosphonium ion to measuredepolarization of macrophages in response to immune complexes,and they found a progressively greater

Annual Reviews

Annu. Rev. Immunol. 1988.6:251-281. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

FCy RECEPTORS

267

depolarizationin responseto complexes of increasingsize. Similar results were obtained with membranevesicles, and the depolarization was assigned to an inward flow ofNa+ (Younget al, 116). MacrophageF%R, purified by affinity chromatography, wasalso reconstitutedin one leaflet of a lipid bilayer spanningan annulus betweentwo compartments.Following application of a voltage across the bilayer, current wasmeasured(117). Whenthe receptor was cross-linked with either the MAb2.4G2or immune complexes, ion channels with a conductance of 60 pS in 1 MKC1were observed. The channels behavedas ohmicconductors, with conductivity 2+ was12 : 1. Studyfor Na+ and K+. Therelative permeability of Na+/Ca ing humanalveolar macrophagesby patch clamping techniques, Nelson et al (118) reported large ion channels induced by aggregated immu+ noglobulin. Thechannelswerecation-specific, withoutselectivity for Na relative to K+, similar to the studies of Young et al (117)in whichpurified FcvRwasincorporatedinto planar lipid bilayers. However,Wilsonet al (119) do not find any depolarization of B cells by F%Rcross-linking, but they report a lower intracellular Ca++ and inositol triphosphateelevationin responseto anti-Is. Therole of Ca+ + in F%R-mediated phagocytosis by macrophagesis controversial. Measuring internal Ca2+by quirt-2 fluorescence, Lewet al (120) found that binding of immune complexesto neutrophils resulted in a slowincrease in internal Ca2+ that occurred with a lag phase. Theseresults were confirmedby Di Virgilio et al (121) in macrophages presented with F%R-ligands.However, wheninternal Ca2 ÷ levels wereloweredby either depletionof intracellular Ca2÷ by ionomycinin the presence of EGTA,or by permeabilizing the 4- in the presence of EGTA,no inhibition of phagomembranewith ATP cytosis wasobserved. Theseresults suggest that the proximalsignal for the F%R triggered phagocyticevent is mostlikely not elevation in internal Ca2+, derived from either internal or external stores. In view of the complexityrevealed in the cloning studies (see below), further study the physiological contributions of each individual receptor are clearly warranted. Macrophagesstimulated with immunecomplexesrelease arachidonic acid (20 : 4) and arachidonatemetabolites. Aderemet al (122) haveexamined the requirementfor Na÷ in release of 20 : 4 frommousemacrophages and find that F%R-mediated release is Na÷-dependent,whereas phorbol ester- and A23187-mediated release of 20 : 4 is not affected by substitution of K+ for Na+. Therequirementfor external Na+ and for protein synthesis can be bypassedwith external Ca2+ and the ionophoreA21387.Pfefferkorn (123) reported that phagocytosisof IgG-sensitizedToxoplasma gondii by J774macrophages wasequally efficient in mediain whichK÷ or choline replaced Na+ as control medium,suggesting that Na+ fluxes per se are

Annual Reviews

Annu. Rev. Immunol. 1988.6:251-281. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

268

UNKELESS, SCIGLIANO & FREEDMAN

not involved in signalling. She proposesinstead an ion-flux independent stimulus operating through Fc~Rconformationalchange. Suzukiand his colleagues havedescribed the purification of Fc~Rfrom both human(124) and mudne(125-128) cell lines by chromatography Sepharosecoupled with a phosphatidylcholine(PC) analog or on aggregated IgG-Sepharose.Fernandez-Botran& Suzuki (127) reported the PCbinding protein had a sharp isoelectric point of 5.8 in 6 Murea and that surface-iodinated proteins isolated by similar methodology (126) had a of 42,000. Theyfound that the PC-bindingprotein inhibited the formation of EIgG2b,but "not EIgG2arosettes with macrophages(126). Furthermore, the protein had a Ca+ 2-dependentphospholipaseA2activity that wasstimulated up to 9-fold in the presenceof IgG2baggregates(126). The signalling mechanismof the moF%R specific for IgG2b aggregates is thereforethoughtto proceedby release of arachidonicacid directly, resulting in conversionto prostaglandins, whichcan then stimulate the adenyl cyclase of the macrophage (125). However,the biochemicalcharacterization of the PC-bindingprotein as moF%RII remains an open question. The yield of PC-bindingprotein fromthe P388D1 cell line is between0.7 to 4 mgper 109 cells. If the size of the Fc~Rreported previously, 42,000kd (126) is correct andone assumes a 100%yield of 0.7 mgof FcrR per 10 9 cells, the numberof FcrRsites wouldhave to be 107 per cell, whichwouldbe greater than 20-fold the numberdetected by binding either IgG ligands or the anti-F%RMAb 2.4G2to macrophagecell lines (79, 80, 82). No SDS-PAGE analysis the unlabeledpurified PC-bindingprotein is presented, but the size and isoelectric point of the surface-labeledmaterialis substantially different fromresults obtained by others (89-93, 129) for moF%RII. Thecalculation for the size of the PC-binding protein as 417residuesrests on an unverified assumptionthat the protein has 15 valine residues. Finally, the aminoacid compositionof the PC-bindingprotein (127) is not consistent with that predicted from the cDNA sequenceof cloned F%R(3, 4, 150). Theobservation that there is stimulation of a phospholipaseA2activity by aggregated IgGis, however,significant becausestimulation ofmacrophage FcrR by immunecomplexesleads to release of arachidonic acid, whichis then convertedinto prostaglandins and leukotrienes. Fernandez-Botran & Suzuki (128) have also examinedthe proteins bound to aggregated IgG-Sepharose.Three major proteins were found, with Mrof 50,000, 25,000, and 17,000. The 50,000 Mr protein could be separated from the smaller proteins by SephadexG-100filtration, and whenincorporated into liposomes,it inhibited the binding of IgG2a-Ebut not IgG2b-Eto macrophages.Theisoelectric point reported by Nitta et al (126) for the IgGbinding protein was4.8, but only a 25,000Mrprotein

Annual Reviews

Annu. Rev. Immunol. 1988.6:251-281. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

FC~.,

RECEPTORS

269

was seen uponSDS-PAGE, in contrast to the later report (128). Suzuki and his colleagues find a role for the IgG-Sepharosebinding proteins in the activation of adenyl cyclase. The25,000and/or 17,000Mrproteins in combinationwith the 50,000 Mrprotein, whenfused into membranesof cells with nonfunctional G protein, could stimulate a twofold IgG2adependent activation of endogenousadenyl cyclase (128). The adenyl cyclase stimulation wasablated by trifluoperazine, implicatingcalmodulin and Ca2+ in the linkage betweenthe FcvRand adenyl cyclase. The possibility that other subunitsmaybe present in an Fc~Rcomplexthat interacts directly with adenylcyclaseis very interesting andwarrantsa moredetailed characterization of the proteins involved. SOLUBLE

FC~R

MOLECULES

Murine Several investigators havedescribed soluble factors present in cell-free culture supernatants or circulating in serumwhichbind immunoglobulin via the Fc fragment(Ig-bindingfactors or IBF). Thesefactors haveexcited a great deal of interest becauseof their putative roles in the delicate feedbackloops of the immunesystem, and they have been the subject of several comprehensive reviews (130). Fridmanand his colleagues (131) first described an immunoglobulin binding factor present on and producedby murineT cells. Subsequently, Gisler & Fridman(132) described a factor, secreted by alloantigen-activated mouseT cells, whichboundIgG. This factor, purified by affinity chromatographyon IgG-coated sepharose columns, directly suppressed the anti-E plaque-forming response of mousespleen cells. Similarly, murineL-5178Ythymoma cells, biosynthetically labelled with radioactive aminoacids, released a radiolabelled factor that boundto IgG-sensitized erythrocytesand also suppressedthe in vitro antibodyresponseto E (133). An FeaR-positive T-cell hybridomaclone, T2D4,isolated by NeauportSautes et al (134) wasshownto secrete an immunoglobulin binding factor that had suppressiveactivity on an in vitro antibodyresponseto E. This factor was induced by treatment of T2D4cells with a monoclonalIgG1 and inhibited rosette formationby spleen cells with IgGl-sensitizedsheep E, but it had no effect on IgG2aor IgG2b-mediatedrosette formation. WhenT2D4cells were incubated with a myelomaIgG2a, factors that specifically inhibited rosette formationwith IgG2a-Eor IgG2b-Ebut not with IgG1-Ewere induced and isolated from the culture supernatants (135). Theauthors concludedfromthese studies that specific IgGisotypes can induceT-cell derived soluble factors that bind immunoglobulin of the samespecificity as the inducing immunoglobulin.

Annual Reviews

Annu. Rev. Immunol. 1988.6:251-281. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

270

UNKELESS, SCIGLIANO & FREEDMAN

The studies described above have concentrated on immunoglobulin bindingfactors derivedfromT-cells. However, it appearsthat these soluble immunoglobulin binding factors maybe derived from a variety of cells both in vitro and in vivo. For example,Loube& Dorrington(136) purified a soluble Fc binding protein from the spent culture fluid of the P388D1 murine macrophageline. Furthermore, murine spleen cells activated in vitro with LPSrelease soluble proteins reactive with the anti-mouseF%R monoclonal antibody, MAb2.4G2, as measured in a sandwich radioimmuneassay (137). TheLPS-inducedFc~Ractivity is associated with cells, since depletionof either adherentcells or T cells fromthe cultures does not changethe expressionof the activity. In addition, pooledserum or plasmafrom various inbred mousestrains contained a similar protein activity, reactive with MAb2.4G2. Analogousresults were reported by Khayatet al (138) whodetected a 2.4G2-reactive molecule in normal mouseserum. This soluble 2.4G2-reactive factor could be removedfrom mouseserumby IgG-Sepharosebut not by Sepharosecoupled with F(ab’)2 fragmentsof IgG. Khayatet al (139) also showedthat the level of the soluble mouseF%Rwas elevated during infection. Whetherthe soluble receptoris the productof a different gene, alternate splicing, or a result of proteolysis of the membrane-bound F%R,remainsto be clarified. The tremendousheterogeneity of the immunoglobulinbinding factors is evident from studies of their structure, whichwererecently reviewed (140, 141). The immunoglobulinbinding factors released by murine 5178Ylymphomacells range from 80,000 to 300,000 Mr, while immunoglobulin binding factors isolated from the supernatant of T2D4cells ranged from 19,000 to 78,000 Mr. The affinity purified immunoglobulin binding factor from T2D4cells is also heterogenousin charge and carbohydrate content (142). Dacronet al (143) have demonstrated,moreover, that at least someof the immunoglobulin binding factors released in the supernatant of the T2D4hybridomabear 2.4G2 determinants, as shown by affinity chromatographyof the cell-free supernatants over 2.4G2Sepharose.These2.4G2-reactiveproteins were capable of binding IgGin the biologic assays used to identify immunoglobulin binding factor, again indicating the structural relationship betweenthe cell-bound membrane receptor and the soluble receptor. To date, cloning workhas not shown multiple T cell FcrR, so that the identity of the multiple IgG binding proteins, as well as their biologicfunction(s) in vivo awaitresolution. To clarify the possible regulatory role of the soluble F%R,Fridmanet al (141) investigated the effect of the T2D4immunoglobulin bindingfactors on hybridomaB-cell lines, each of which secreted IgG of different subclasses. T2D4T-hybridoma-derivedimmunoglobulinbinding factor (IgG-BF)was purified by affinity chromatographyon IgG-Sepharoseand

Annual Reviews FCr RECEPTORS

271

then incubated with the monoclonalB cells. They report that IgG-BF suppressed antibody production in a dose- and time-dependent manner. Furthermore,IgG-BFappears to exert a cytostatic effect and inhibits proliferation of the B cells, although the mechanism of this effect is unknown(144, 145).

Annu. Rev. Immunol. 1988.6:251-281. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Human Severaldifferent humancell types havebeenfoundto release a soluble Fc~R in serum-free medium.U937cells, which bear huFc~RIand huF%RII, spontaneouslyrelease a factor whichis retained on Sepharose-IgG but not on Sepharose-F(ab’)2-IgG (146). This factor inhibits IgGsynthesis in vitro by pokeweedmitogen stimulated B cells. In addition, normal human peripheral mononuclearcells shed a soluble FcrR whichcan be further purified by affinity chromatographyon IgG-Sepharosecolumns or on concanavalin A-Sepharosecolumns (147). This soluble receptor also inhibits secondaryantibodyproductionin vitro. Functionally active F%Rwith a Mr of 60,000 on SDS-PAGE has also been isolated from normalhumanserumby affinity chromatography (148). Recently, a sandwichELISAassay has been developedto detect soluble FcRin normalhumanserum(149). This assay utilizes monoclonalantibodies3G8and anti-leu-1 lb, whichrecognizetwodistinct epitopes on the receptor, and has been employedto demonstratethe presence of CD16reactive moleculesin humanserum. Thesize, composition,and biological function of these moleculeshave not been determined. The purification and cloning of the humansoluble receptors will promotean understanding of their role(s) in both normaland pathologicconditions.

CLONING OF Fc~R GENES The recent cloning of murineF%Rsby three groupsshould lead to rapid progress in elucidation of the relationship betweenstructure and function for these importantmolecules.Theresults fromthe different laboratories are in substantial agreement (Ravetchet al, 3; Lewiset al, 4; Hibbset al, 5; Hogarthet al, 150). The F%R is another memberof the immunoglobulin gene superfamily, with closest homologyto class II histocompatibility antigens. Followinga leader sequencethere is an extracellular portion that consists of two repeated immunoglobulin-likedomains. Eachdomain,of approximately85 aminoacids, has two sites for N-linkedoligosaccharide, and two cysteine residues, whichare separated by 42 to 45 aminoacids. This is in agreementwith the experimentalevidenceof Greenet al (151), demonstrating 4 N-linked glycosylation sites. The external repeated domains showhomologyto immunoglobulins,MHC class I and II prod-

Annual Reviews

Annu. Rev. Immunol. 1988.6:251-281. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

272

LrNKELESS,

SCIGLIANO & FREEDMAN

ucts, f12 microglobulin, and other membersof the immunoglobulin supergene family. The most striking homology,however,is to the f12 domain of the Eft class II gene, with 32%homology over a 91 aminoacid stretch. The Fc~Rhas a single transmembranehydrophobic domainand a cytoplasmic domain. Themethodused by Ravetchet al (3) to clone the F%R wasto construct an oligonucleotideprobe basedon Nterminal sequenceof protein purified by affinity chromatography on MAb2.4G2-Sepharose. This method resulted in the isolation of two F%Rgenes, termed ~ and ft. These two genes show95%homologywithin the extracellular immunoglobulin-like domainbut showa completelack of homology in the leader sequences, the transmembraneand cytoplasmic domains, and in the 3’ and 5’ noncoding region of the cDNA.The ~ gene transcript is found only in macrophage cell lines and wasnot detected in any Fc~Rpositive B cell or T cell line examined,whereasthe fl geneis foundin all F%R positive lines examined to date. It is clear fromtransfection experimentsthat the fl geneencodesthe 2.4G2MAbdeterminant, since cloning of the fl cDNA into the pcEXV-3 eukaryotic expression vector followed by transfection into a mouse melanomacell resulted in expression of F%Rinhibited by MAb2.4G2. Also, Lewiset al (4) find that a B cell mutantlacking FcrRalso lacks the fl genetranscript. Thefunction of the 0~ geneis understudy. Since it is macrophage specific, the identification of the ~ geneproduct as the high avidity moF%RI is an attractive hypothesis. In additionto the presenceof twodifferent genes,Ravetchet al (3) also foundevidencefor differential mRNA processing. Twofl genetranscripts werefound, identical save for a 138-nucleotideinsertion in the cytoplasmic domain.The 138-nucleotide insertion would code for an additional 46 aminoacids, and tryptic fragmentswith sequencein this area werein fact found, ruling out a cloning artifact. In addition, ribonucleaseprotection experimentsdemonstratedboth transcripts in all F%Rpositive B and T cell lines, but onlythe smallerof the twofl transcripts (f12) in macrophages. The functions of the transmembraneand cytoplasmic domainsof the ~ and fl receptors are not understood,but it wouldseemlikely that, given the striking differences, there are also differencesin the types of signals transmittedto the cell. Thesignificanceof the insertion of 46 aminoacids in the fll comparedto the/32 transcripts is also not understood.However, one mayexpect rapid progress in all these areas with the availability of the cloned genesand the ability to transfect them, or mutationsthereof, into variouscell types. The0~ chain of the high avidity rat mastcell/basophil receptor for IgE (rtFc,RI) has recently been cloned by Kinet et al (151) from the basophilic leukemiaRBL.The rtFc~RI is a receptor of exceedingly high

Annual Reviews

Annu. Rev. Immunol. 1988.6:251-281. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

FC~, RECEPTORS

273

avidity and has been thoroughly characterized--see Metzgeret al (152) for review. Thereceptor has four subunits, one ~ chain with the binding site for IgE, a fl chain, and two disulfide-linked ~ chains. Thesequence predicted from the eDNAclone shows pronounced homologywith the moFc~R clones discussed above(3-5). Theoverall identity is 32%between the two genes, and at the nucleic acid level is 49%.The~ subunit of the raFc~RIhas a 180 aminoacid extracellular domain,whichhas two domains in whichthe positions of the cysteines are conserved. There are seven potential N-linkedglycosylationsites. ThertFc~RI~ chain has a 20-residue transmembranedomainand a 27-residue cytoplasmic domain, which has 9 basic aminoacids. Twoareas showextremehomology:l0 of 14 residues in the N-terminusare identical, and 8 aminoacids surroundingthe aspartic acid residing in the transmembrane domainare identical to the sequence predicted for the moFc~R(~) transcript (3). Thepresenceof a negativelychargedaspartic acid residue in the transmembranedomains of both the ~ chain of the rtFc~RI and moF%R(~) chain is unusualand maybear on the difficulty that Kinet et al (146) and J. Ravetch(personal communication)had in expressing the cloned genes in eukaryotic cells. Since it is knownthat the rtFc~RI is composedof multiple subunits, the difficulty in expressingthe protein maybe related to the requirementfor cosynthesisof the other subunitsfor expressionon the cell surface, similar to the requirementsfor /32 microglobulinfor MHC class I antigen expressionand the/3 chain for expression of the ~/3 heterodimersof the CR3/LFA-1/p150,95 family of cell surface receptors. The homologybetween the rtFc,RI ~ chain and the moFcrR(~)sequence in the transmembranedomainalso suggests that other subunits maybe associated with the moF%R(~) peptide to form a functional receptor.

FUTURE

PROSPECTS

Detailed study of the interaction of F%R,a member of the immunoglobulin gene superfamily, with IgG mayprovide a fascinating glimpse into the parallel evolution of ligand/receptor systems. HumanF%Rsare being clonedby several groups, and the preliminaryresults are largely parallel to the murinesystem. Wecan look forwardto studies on the regulation, at the genetic level, of the expressionof FcrRs,whichare underdevelopmentaland hormonalcontrol. Anotherarea in whichthere should be rapid progressis in the delineation of function for individual receptors and the signalling mechanism(s) involved. Cloningstudies and biochemicalstudies of soluble FcrRs, or immunoglobulin bindingfactors, will lead to a better understandingof the immunoregulatory role of these molecules.

Annual Reviews 274 UNKELESS,SCIGLIANO& FREEDMAN ACKNOWLEDGMENT This work was supported by PHSAI24322 and HL38498.

Annu. Rev. Immunol. 1988.6:251-281. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Literature Cited 1. Wright, A. E., Douglas, S. R. 1903. An experimental investigation of the role of the body fluids in connection with phagocytosis. Proc. R. Soc. London72: 357-20 2. Berken, A., Benacerraf, B. 1966. Properties of antibodies cytophilic for macrophages. J. Exp. Med. 123:1193. Ravetch, J. V., Luster, A. D., Weinshank, R., Kochan, J., Pavlovec, A., Portnoy, D. A., Hulmes,J., Pan, Y. C., Unkeless, J. C. 1986. Structural heterogene.ity and functional domains of munne immunoglobulin G Fc receptors. Science 234:718-25 4. Lewis, V. A., Koch, R., Plutner, H., Mellman, I. 1986. A complementary DNAclone for a macrophage-lymphocytc Fc receptor. Nature 324: 37275 5. Hibbs, M. L., Walker, I. D., Kirszbaum, L., Pietersz, G. A., Deacon, N. J., Chambers, G. W., McKenzie, I. F., Hogarth, P. M. 1986. The murine Fc receptor for immunoglobulin purification, partial amino acid sequence, and isolation of eDNAclones. Proc. Natl. Aead. Sci. USA 83:6980-84 6. Unkeless, J. C., Fleit, H., Mellman, I. S. 1981. Structural aspects and heterogeneity of immunoglobulin Fc receptors. Adv. Immunol. 31:247-70 7. Dickler, H. B. 1976. Lymphocyte receptors for immunoglobulin. Adv. lmmunol. 24:167-14 8. Huber, H., Fudcnberg, H. H. 1970. The interaction of monocytes and macrophages with immunoglobulins and complement. Ser. Haematol. 3:160-75 9. Huber, H., Douglas, S. D., Nusbacher, J., Kochwa,S., Rosenfield, R. E. 1971. IgG subclass specificity of human monocyte receptor sites. Nature 229: 419-20 10. Anderson, C. L., Abraham,G. N. 1980. Characterization of the Fc receptor for IgG on a humanmacrophage cell line, U937. J. Immunol. 125:2735-41 11. Anderson, C. L. 1982. Isolation of the receptor for IgG from a human monocyte cell line (U937) and from human peripheral blood monocytes. J. Exp. Med. 156:1794-1806

12. Anderson, C. L., Spence, J. M., Edwards, T. S., Nusbacher, J. 1985. Characterization of a polyvalent antibody directed against the IgG Fc receptor of humanmononuclear phagocytes. £ Immunol. 134:465-70 13. Anderson, C. L., Guyre, P. M., Whitin, J. C., Ryan, D. H., Looney, R. J., Fanger, M. W. 1986. Monoclonal antibodies to Fc receptors for IgG on human mononuclear phagocytes. Antibody characterization and induction of superoxide production in a monocyte cell line. d. Biol. Chem.261:12856~4 14. Frey, J., Engelhardt, W. 1987. Characterization and structural analysis of Fc~ receptors of human monocytes, a monoblast cell line (U937)and a myeloblast cell line (HL-60) by a monoclonal antibody. Eur. d. Immunol. 17:583-91 15. O’Grady, J. H., Looney, R, J., Anderson, C, L. 1986. The valence for ligand of the human mononuclear phagocyte 72 kD high-affinity IgG Fc receptor is one. J. Immunol. 137:2307-10 16. Schreiber, R. D. 1984. Identification of gamma-interferon as a murine macrophage-activating factor for tumor cytotoxicity. Contemp. Top. Immunobiol. 13:171-98 17. Guyre, P. M., Morganelli, P. M., Miller, R. 1983. Recombinant immune interferon increases immunoglobulin G Fe receptors on cultured humanmononuclear phagocytes. J. Clin. Invest. 72: 393-97 18. Akiyama, Y., Lubeck, M. D., Steplewski, Z., Koprowski, H. 1984. Induction of mouse IgG2a- and IgG3-dependent cellular cytotoxicity in humanmonocytic cells (U937) by immune interferon. Cancer Res. 44:5127-31 19. Shen, L., Guyre, P. M., Anderson, C. L., Fanger, M. W. 1986. Heteroantibody-mediated cytotoxicity: antibody to the high affinity Fc receptor for IgG mediates cytotoxicity by humanmonocytes that is enhanced by interferon-gamma and is not blocked by human IgG. J. ImmunoL137: 337882 20. Graziano, R. F., Fanger, M. W. 1987. Human monoeyte-mediated cytotoxiclty: The use of Ig-bearing hybrid-

Annual Reviews

Annu. Rev. Immunol. 1988.6:251-281. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

FCr RECEPTORS

275

W., Capel,P. J,, Koene,R. A. 1984.Fc omasas target cells to detect trigger receptors for mouse IgG1 on human molecules on the monocyte cell surface. J. lmmunol.138:945-50 monocytes: polymorphism and role in atxtibody-iBduced T cell proliferation. 20a. Shen, L., Guyre, P. M., Fanger, J. Immunol.133:1185-89 M. W.1987. Polymorphonuclear leukocyte function triggered through the 31. Clement,L. T., Tilden, A. B., Dunlap, N. E. 1985. Analysis of the monocyte high affinity Fc receptor for monomeric Fc receptors and antibody-mediated IgG. J. Immunol.139:534-38 21. Girard, M.T., Hjaltadottir, S., Fejescellular interactions required for the Toth, A. N., Guyre,P. M. 1987. Gluinduction of T cell proliferation by cocorticoids enhancethe gamma-interanti-T3 antibodies. J. Immunol.135: feron augmentation of humanmono165-71 cyte immunoglobulinG Fc receptor 32. Looney,R. J., Abraham,G. N., Anderexpression. J. lmmunol.138:3235-41 son, C. L. 1986. Humanmonocytesand 22. Yancey,K. B., O’Shea,J., Chused,T., U937cells bear two distinct Fc receptors for IgG. J. Immunol.136:1641-47 Brown,E., Takahashi, T., Frank, M. 33. Rosenfeld,S. I., Ryan,D. H., Looney, M., Lawley, T. J. 1985. HumanC5a modulatesmonocyteFc and C3 recepR. J., Anderson, C. L., Abraham,G. N., Leddy, J. P. 1987. HumanFc tor expression. J. ImmunoL 135: 46570 gammareceptors: Stable inter-donor variation in quantitative expressionon 23. Rosenfeld,S. I., Looney,R. J., Leddy, J. P., Phipps, D. C., Abraham,G. N., platelets correlates with functional Anderson,C. L. 1985. Humanplatelet responses. J. Immunol.138:2869-73 Fc receptor for immunoglobulinG. 34. Per, A., Looney,R. J., Ryan,D. H., Identification as a 40,000-molecularAbraham,G. N. 1986. Thelow affinity weight membraneprotein shared by 40,000 Fc gammareceptor and the monocytes.J. Clin. Invest. 76:2317-22 transferrin receptor can be alternative 24. Kulczycki, A. Jr. 1984. Humanneuor simultaneoustarget structures on trophils and eosinophils have struccells sensitive for natural killing. J. turally distinct Fc gamma receptors. J. Immunol. 136:4714-20 Immunol. 133:849-54 35. Vodinelich,L., Sutherland,R., Schnei25. Looney,R. J., Ryan,D. H., Takahashi, der, C., Newman,R., Greaves, M. 1983. Receptorfor transferrin maybe K., Fleit, H. B., Cohen, H. J., Abraham, G. N., Anderson,C. L. 1986. a "target" structure for natural killer Identification of a secondclass of IgG cells. Proc. Natl. Acad.Sci. USA80: Fc receptors on humanneutrophils. A 835-39 40 kilodalton moleculealso foundon 36. Alarcon,B., Fresno,M. 1985.Specific eosinophils. J. Exp. Med.163:826-36 effect of anti-transferrin antibodieson 26. Jones, D. H., Looney,R. J., Anderson, natural killer cells directed against C. L. 1985.Twodistinct classes of IgG tumor cells. Evidencefor the transFc receptors on a humanmonocyteline ferrin receptor beingoneof the target structures recognizedby NKcells. J. (U937)defined by differences in binding of murine IgG subclasses at low Immunol. 134:1286-91 ionic strength. J. lmmunol.135: 3348- 37. Werner, G., von demBorne, A. E. G. 53 Kr., Bos, M. J. E., Tromp,J. F., van 27. McCool,D., Birshtein, B. K., Painter, der Plas-van Dalen, C. M., Visser, F. R. H. 1985. Structural requirementsof J., Engelfdet,C. P., Tetteroo,P. A.T. immunoglobulinG for binding to the 1986. Localization of the humanNA1 Fc gammareceptors of the human alloantigen on neutrophil Fc-), receptors. In LeukocyteTypin#II, Vol. 3. tumor cell lines U937,HL-60,ML-1, and K562. J. Immunol.135:1975-80 HumanMyeloid and Hematopoietic 28. Anderson,C. L., Ryan,D. H., Looney, Cells, ed E. L. Reinherz,B. F. Haynes, R. J., Leary, P. C. 1987. Structural L. M. Nadler,I. D. Bernstein,pp. 109polymorphismof the humanmonocyte 21. NewYork: Springer-Verlag 40 kilodalton Fc receptor for IgG. J. 38. Fleit, H.B., Wright,S. D., Unkeless,J. Immunol.In press C. 1982. Humanneutrophil Fc gamma 29. Tax, W.J., Willems,H. W., Reekers, receptor distribution and structure. P. P., Capel,P. J., Koene,R. A. 1983. Proc. Natl. Acad.Sci. USA79’. 3275Polymorphismin mitogenic effect of 79 IgG1 monoclonalantibodies against 39. Perussia, B., Starr, S., Abraham,S., T3 antigen on humanT ceils. Nature Fanning, V., Trinchieri, G. 1983. 304:44.%47 Human natural killer cells analyzedby 30. Tax, W.J., Hermes,F. F., Willems,R. B73.1, a monoclonalantibody blocking

Annual Reviews

Annu. Rev. Immunol. 1988.6:251-281. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

276

UNKELESS, SCIGLIANO & FREEDMAN

Fc receptor functions. I. Characterization of the lymphocytesubset reactive with B73.1. J. Irnmunol.130: 2133-41 40. Perussia, B., Acuto,O., Terhorst, C., Faust, J., Lazarus, R., Fanning, V., Trinchieri, G. 1983. Humannatural killer cells analyzedbyB73.I, a monoclonal antibody blocking Fc receptor functions. II. Studies of B73.1antibody-antigen interaction on the lymphocyte membrane.J. Immunol. 130: 2142-48 41. Perussia, B., Trinchieri, G., Jackson, A., Warner,N. L., Faust, J., Rumpold, H., Kraft, D., Lanier, L. L. 1984. The Fc receptor for IgGon humannatural killer cells: phenotypic,functional,and comparative studies with monoelonal antibodies. J. Immunol.133:180--89 42. Perussia, B., Trinchieri, G.1984.Antibody3G8,specific for the humanneutrophil Fc receptor, reacts with natural killer cells. J. Immunol.132:1410-15 43. Fleit, H. B., Wright,S. D., Durie, C. J., Valinsky,J. E., Unkeless,J. C. 1984. Ontogeny of Fc receptors and complement receptor (CR3)during human myeloiddifferentiation.J. Clin. Invest. 73:516-25 44. Madyastha,P. R., Fudenberg,H. H., Glassman, A. B., Madyastha,K. R., Smith, C. L. 1982. Autoimmune neutropeniain early infancy:a review.Ann. Clin. Lab. Sci. 12:356-67 45. Lalezari, P., Khorshidi,M., Petrosova, M. 1986. Autoimmune neutropenia of infancy. J. Pediatr. 109:764-69 46. Tetteroo, P. A. T., van der Schoot,C. E., Visser,F. J., Bos,M.J. E., yondem Borne, A. E. G. Kr. 1987. Three different types of F%receptors on humanleukocytes defined by workshopantibodies: F%Rto of neutrophils, Fe~R~o of K/NKlymphocytes and Fc~RII.In LeukocyteTypingIli, Vol. 3, HumanMyeloid and Hematopoietic Cells, edited by A. MeMiehael, Oxford Univ.Press. In press 47. Clarkson, S. B., Ory, P. A. 1987. CD16: Developmentallyregulated IgGFc receptors on cultured monocytes. J. Exp. Med.In press 48. Kurlander,R. J., Barker, J. 1982. The binding of humanimmunoglobulinG1 monomer and small, covalently crosslinked polymers of immunoglobulin G1 to humanperipheral blood monocytes and polymorphonuclearleukocytes. J. Clin. Invest. 69:1-8 49. Kurlander, R. J., Haney, A. F., Gartrell, J. 1984. Humanperitoneal macrophagespossess two populations

of IgGFc receptors. Cell Immunol.86: 479-90 50. Clarkson,S. B., Kimberly, R. P., Valinsky, J. E., Witmer,M.D., Bussel,J. B., Nachman, R. L., Unkeless,J. C. 1986. Blockadeof clearance of immunecomplexes by an anti-Fc gammareceptor monodonal antibody. J. Exp. Med. 164:474-89 51. Frank, M.M., Hamburger,M. I., Lawley, T. J., Kimberly,R. P., Plotz, P. H. 1979. Defectivereticuloendothelial systemFc receptor functionin systemic lupus erythematosus.N. EngLJ. Med. 300:518-20 52. Anasetti, C., Martin, P. J., Modshita, Y., Badger, C. C., Bernstein, I. D., Hansen, J. A. 1987. Humanlarge granular lymphoeytes express high affinity receptors for murine monoclonal antibodies of the IgG3 subclass. J. lrnmunoL138:2979-81 P., Punt,J., Her53. Kipps,T. J., P~rham, zenberg, L. A. 1985. Importance of immunoglobulin isotype in human antibody-dependent,cell-mediated cytotoxicity directed by routine monoclonal antibodies. J. Exp. Med.161: 117 54. Christiaansen, J. E., Burnside,S. S., Sears, D. W.1987.Apparentsensitivity of humanK lymphocytesto the spatial orientation and organizationof target cell-bound antibodies as measuredby the efficiency of antibody-dependent cellular cytotoxicity (ADCC). J. Imraunol. 138:2236-43 55. Titus, J. A., Perez, P., Kaubisch,A., Garrido, M. A., Segal, D. M. 1987. HumanK/NKcells targeted with heterocrosslinkedantibodies specifically lyse tumorcells in vitro and prevent tumor growthin vivo. J. Immunol.In press 56. Griffin, J. A., Griffin, F. M.Jr. 1979. Augmentation of macrophage complementreceptor function in vitro. I. Characterizationof the cellular interactions required for the generationof a T-lymphocyteproduct that enhances macrophage complement receptor function. J. Exp. Med.150:653-75 57. Griffin, F. M.Jr., Griffin, J. A. 1980. Augmentation of macrophage complementreceptor function in vitro. II. Characterization of the effects of a unique lymphokine upon the phagocytic capabilities of macrophages.J. Immunol. 125:844-49 58. Gresham, H. D., Clement, L. T., Lehmeyer,J. E., Griffin, F. M. Jr., Volonakis, J. E. 1986. Stimulation of human neutrophil Fc receptor-

Annu. Rev. Immunol. 1988.6:251-281. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Annual Reviews

mediatedphagocytosisby a low molecular weightcytokine. J. Immunol.137: 868-75 59. Jack, R. M., Fearon, D. T. 1984. Altered surface distribution of both C3b receptors and Fc receptors on neutrophils inducedby anti-C3b receptor or aggregated IgG. J. lmmunol.132: 3028-33 60. Lawley,T. J., Hall, R. P., Fauci,A.S., Katz, S. I., Hamburger,M. I., Frank, M. M. 1981. Defective Fc-receptor functions associated with the HLAB8/DRw3 haplotype: studies in patients with dermatitis herpetiformis and normalsubjects. N. EngLJ. Med. 304:185-92 61. Salmon,J. E., Kimberley,R. P., Gibofsky, A., Fotino, M. 1986. Altered phagocytosis by monocytes from HLA-DR2 and DR3-positive healthy adults is Fc gamma receptorspecific. J. lmmunoL136:3625-30 62. Salmon,J. E., Kimberley,R. P. 1986. Phagocytosis of concanavalin A-treated erythrocytesis mediatedby the Fc gammareceptor. J. lmmunoL 137: 45662 63. Lawley,T. J. 1980. Immune complexes and reticuloendothelial system function in humandisease. J. Invest. DermatoL74:339-43 64. Salmon,J. E., Kimberley, R. P., Gibofsky, A., Fotino, M. 1984. Defective mononuelear phagocyte function in systemic lupus erythematosus: dissociation of Fc receptor-ligandbinding and internalization. J. lmmunoL 133: 2525-31 65. Bussel, J. B. 1986. Intravenousimmunoglobulintherapyfor the treatmentof idiopathic thrombocytopenicpurpura. Prog. Hemost.Thromb.8:103-25 66. Schreiber, A. D., Chien, P., Tomaski, A., Cines,D. B. 1987. Effect ofdanazol in immunethrombocytopenicpurpura. N. Enfl. J. Med.316:503-8 67. Salmon,J. E., Kapur,S., Kimberly,R. P. 1987. Gammaglobulinfor intravenoususe induces an Fc gamma receptor-specific decrementin phagocytosis by blood monocytes. Clin. ImmunoL Immunopathol.43:23-33 68. Clarkson,S. B., Bussel,J. B., Kimbedy, R. P., Valinsky, J. E., Nachman,R. L., Unkeless,J. C. 1986. Treatmentof refractory immunethrombocytopenic purpura with an anti-Fc gamma-receptor antibody. N. En#l. J. Med. 314: 1236--39 69. Ehlenberger, A, G., Nussenzweig,V. 1977. Therole of membrane receptors for C3band C3din phagocytosis. J.

FCr RECEPTORS 277 Exp. Med. 145:357-71 70. Loos, M. 1982. The functions of endogenousClq, a subc, omponentof the first componentof complement, as a receptor on the membraneof macrophages.Mol. Immunol.19: 122938 71. Heinz, H. P., Dlugonska,H., Rude,E., Loos, M. 1984. Monoclonal antimousemacrophageantibodies recognize the globular portions of Clq, a suboomponentof the first component of complement.Z Immunol.133: 40072. Bobak,D. A., Gaither, T. A., Frank, M.M., Tenner, A. J. 1987. Modulation of FcRfunetion by complement:SubcomponentClq enhances the phagoeytosis of IgG-opsonizedtargets by humanmonocytesand calture-derived macrophages.J. Immunol.138: 115056 73. Mocharla, R., Mochada, H., Leu, R. W.1987. Effects of inhibitors of Clq biosynthesis on macrophageFc receptor subclass-mediatedantibodydependentcellular cytotoxicity and phagocytosis.Cell. Immunol.105: 12735 74. Hamada,A., Greene, B. M. 1987. Clq enhancementof IgG-dependenteosinophil-mediatedkilling of Schistosomula in vitro. J. lmmunol.138:1240-45 75. Mentzer, S. J., Barbosa, J. A., Burakoff, S. J. 1985.T3monoelonal antibodyactivationof nonspecificcytolysis: a mechanismof CTLinhibition. J. lmmunoL135:34-38 76. Spits, H., Yssel, H., Leeuwenberg, J., de Bries, J. E. 1985. Antigenspecific cytotoxic T cell and antigen-specific proliferating T cell clones can be inducedto cytolytic activity by monoclonal antibodies against T3. Eur. J. Immugol. 15:88 77. Leeuwenberg, J. F., Spits, H., Tax, W. J., Capel,P. J. 1985.Inductionof nonspecific cytotoxicity by monoclonal anti-T3 antibodies. J. Immunol.134: 3770-75 78. Egawa,S. E., Abo,T., Hiwatashi, N. 1987. Enhancementof humannatural killer activity by the monoclonal Leu11 antibodies.Cell. Imrnunol.104:38699 79. Unkeless, J. C., Eisen, H. N. 1975. Binding of monomeric immunoglobulin to Fc receptors of mouse macrophages.J. Exp. Med.142: 152033 80. Unkeless,J. C. 1977. The presence of two Fc receptors on mouse macrophages:evidencefroma variantcell line

Annual Reviews

Annu. Rev. Immunol. 1988.6:251-281. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

278

UNKELESS, SCIGLIANO & FREEDMAN

anddifferential trypsin sensitivity. J. antigenLy-rn20.2,a cell surfacemarker Exp. Med. 145:931-45 controlled by a gene linked to the 81. Diamond,B., Scharff, M. D. 1980. (Mlslocus.). J. Immunol.135:2635-41 IgG1and IgG2bshare the Fc receptor 94. Nose,M., Wigzell,H. 1983. Biological on mouse macrophages. J. ImmunoL significance of carbohydrate chains 125:631-33 on monoclonalantibodies. Proc. Natl. 82. Unkeless,J. C. 1979. Characterization Acad. Sci. USA80:6632-36 of a monoclonal antibody directed 95. Leatherbarrow,R. J., Rademacher,T. against mouse macrophageand lymW., Dwek,R. A., Woof,J. M., Clark, phocyteFc receptors. J. Exp. Med.150: A., Burton, D. R., Richardson, N., 580-96 Feinstein, A. 1985. Effector functions 83. Diamond,B., Yelton, D. E, 1981. A of a monoclonalaglycosylated mouse new Fc receptor on mouse macroIgG2a: binding and activation of phagesbindingIgG3.J. Exp. Med.153: complementcomponentC1 and inter514-19 action with humanmonocyteFc recep84. Lane,B. C., Bricker, M.D., Cooper,S. tor. Mol. lmmunol.22:407-15 M. 1982. Fc receptors of mousecell 96. Diamond, B., Boecumini,L., Birshtein, B. K. 1985. Site of binding of IgG2b lines. II. IgGbinding specificity and identification of the Fc receptor on a and IgG2a by mouse macrophage Fc lymphoidleukemia. J. lmmunol.128: receptors by using cyanogenbromide 1825-31 fragments. J. Immunol.134:1080-83 85. Phillips, N. E., Parker,D.C. 1985.Sub- 97. Birshtein, B. K., Campbell, R., class specificity of Fc gamma receptorDiamond,B. 1982. Effects of immumediated inhibition of mouseB cell noglobulin(structure) on (Fc receptor) activation. J. Immunol. 134: 2835binding: a mouse myelomavariant 38 immunoglobulin with a gamma2bgamma2a hybrid heavy chain having a 86. Teillaud, J. L., Diamond, B., Pollock, R. R., Fajtova,V., Scharff, M.D. 1985. completegamma 2a (Fc) region fails Fc receptors on cultured myeloma bind to gamma2a (Fc) receptors and hybridomacells. J. Immunol.134: mousemacrophages.J. Immunol.129: 610-14 1774-79 87. Baum,C. M., McKearn,J. P., Riblet, 98. Diamond, B., Boccumini,L., Birshtein, B. K. 1985. Site of binding of IgG2b R., Davie, J. M. 1985. Polymorphism of Fc receptor on murineBcells is Ighand IgG2a by mouse macrophage Fc linked. J. Exp. Med.162:282-96 receptors by using cyanogenbromide 88. Kulczykci, A. Jr. 1984. Humanneufragments.J. Irnrnunol. 134:1080-83 trophils and eosinophils have struc99. Mellman,I. S., Plutner, H., Steinman, turally distinct Fc gamma receptors. J. R. M., Unkeless, J. C., Cohn,Z. A. Immunol. 133:849-54 1983. Internalization and degradation 89. Mellman,I. S., Unkeless, J. C. 1980. of macrophageFc receptors during Purification of a functional mouseFc receptor-mediated phagocytosis. J. receptor through the use of a monoCell Biol. 96:887-95 clonal antibody. J. Exp. Med. 152: 100. Kurlander,R. J., Gartrell, J. E. 1983. 1048~59 The binding and processing of mono90. Lane, B. C., Cooper, S. M. 1982. Fc clonal humanIgG1by cells of a human receptorsof mouse cell lines. I. Distinct macrophage-like cell line (U937).Blood proteins mediate the IgG subclass62:652~2 specific Fc bindingactivities of macro- 101. Jones, D. H., Nusbacher,J., Anderson, phages. J. Irnmunol.128:1819-24 C. L. 1985. Fc receptor-mediatedbinding and endocytosis by humanmono91. Holmes,K. L., Palfree; R. G., Hamnuclear phagocytes: monomeric IgG is merling, U., Morse, H. C. 1II. 1985. not endocytosed by U937cells and Alleles of the Ly-17alloantigen define polymorphismsof the murine IgG Fc monocyteS.J. Cell Biol. 100:558~64 receptor. Proc. Natl. Acad. Sci. USA 102. Ukkonen,P., Lewis, V., Marsh, M., 82:7706-10 Helenius,A., Mellman,I. 1986. Transport of macrophageFc receptors and 92. Hibbs, M. L., Hogarth, P. M., Fc receptor-bound ligands to lysoMcKenzie, I. F. 1985. The mouse Ly-17locus identifies a polymorphism somes.J. Exp. Med.163:952-71 of the Fc receptor. Immuno#enetics 22: 103. Mellman,I., Plutner, H., Ukkonen,P. 1984. Internalization and rapid recy335-48 cling of macrophage Fc receptors 93. Mark, W. H., Kimura, S., Hammerling, U. 1985. Biochemical charactagged with monovalentantireceptor terization of murine lymphoidalloantibody: possible role of a prelyso-

Annual Reviews

Annu. Rev. Immunol. 1988.6:251-281. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

FC~ RECEPTORS somal compartment.J. Cell Biol. 98: 1163-69 104. Mellman,I., Plutner, H. 1984. Internalization and degradation of macrophage Fc receptors bound to polyvalent immune complexes.J. Cell Biol. 98:1170-77 105. Kurlander,R. J., Ellison, D. M., Hall, J. 1984. Theblockadeof Fe receptormediated clearance of immunecomplexes in vivo by a monoclonalantibody(2.4G2)directed against Fc receptors on murineleukocytes. J. Immunol. 133:855-62 106. Kurlander, R., Hall, J. 1986. Comparison of intravenousgamma globulin and a monoclonal anti-Fc receptor antibodyas inhibitors of immune clearancein vivoin mice.J. Clin.Invest. 77: 2010-18 107. Ralph, P., Nakoinz,I., Diamond,B., Yelton, D. 1980. All classes of murine (IgG) antibody mediate macrophage phagocytosisandlysis of erythroeytes. J. Immunol.125:1885-88 108. Matthews, T. J., Collins,J. J., Roloson, G. J., Thiel, H. J., Bolognesi,D. P. 1981. Immunologiccontrol of the ascites form ofmurineadenocarcinoma 755. IV. Characterization of the protective antibody in hyperimmune serum. J. Immunol.126:2332-36 109. Herlyn, D., Koprowski, H. 1982. IgG2amonoclonalantibodies inhibit humantumor growth through interaction with effector ceils. Proc. Natl. Acad. Sci. USA79:4761-65 110. Langiois, A. J., Matthews, T. J., Weinhild,K. J., Bolognesi,D. P. 1985. Immunologiccontrol of a retrovirus associated murine adenocarcinoma. VII. Tumorcell destruction by macrophagesand IgG2a.J. Natl. Can. Inst. 75:709-15 111. Nathan, C., Brukner, L., Kaplan, G., Unkeless, J., Cohn,Z. 1980. Role of activated macrophagesin antibodydependentlysis of tumorcells. J. Exp. Med. 152:183-97 112. Phillips, N.E., Parker,D. C. 1983.Fcdependentinhibition of mouseB cell activation by wholeanti-muantibodies. J. Immunol.130:602-06 113. Phillips, N. E., Parker, D. C. 1984. Cross-linking of B (lymphocyte) gamma receptors and membrane immunoglobulininhibits anti-immunoglobulin-inducedblastogenesis. J. Immunol. 132:627-32 114. Phillips, N.E., Parker,D.C. 1985.Subclass specificity of Fc gamma receptormediatedinhibition of mouseB cell activation. J. Immunol.134:2835-38

279

115. Young,J. D., Unkeless,J. C., Kaback, H. R., Cohn,Z. A. 1983. Macrophage membranepotential changes associated with gamma 2b/gamma 1 Fc receptor-ligand binding. Proc. Natl. Acad. Sci. USA80:1357-61 116. Young,J. D., Unkeless, J. C., Young, T. M., Mauro,A., Cohn, Z. A. 1983. Mouse macrophage Fc receptor for IgG gamma2b/gamma1 in artificial and plasma membranevesicles functions as a ligand-dependentionophore. Proc. Natl. Acad.Sci. USA80: 1636-40 117. Young,J. D., Unkeless,J. C., Young, T. M., Mauro,A., Cohn, Z. A. 1983. Role for mouse macrophage IgG Fc receptor as ligand-dependention channel. Nature 306:186-89 118. Nelson,D. J., Jacobs, E. R., Tang,J. M., Zeller, J. M., Bone, R. C. 1985. Immunoglobulin G-induced single ionic channels in human alveolar maerophagemembranes.J. Clin. Invest. 76:500~7 119. Wilson,H. A., Greenblatt, D., Taylor, C. W.,Putney,J. W.,Tsien, R. Y., Finkelman, F. D., Chused, T. M. 1987. The B lymphocytecalcium response to anti-Ig is diminished by membrane immunoglobulincross-linkage to the Fc gammareceptor. J. Immunol.138: 1712-18 120. Lew,D. P., Andersson,T., Hed,J., Di Virgilio, F., Pozzan,T., Stendahl,O. 1985. Caz+ -dependent and Ca2+-inde pendent phagocytosis in humanneutrophils. Nature315:509-11 121. Di Virgilio, F., Meyer, B. C., Silverstein, S. C. 1987. Fc receptormediated phagocytosis occurs in macrophagesat exceedingly low cytosolic Ca2+levels. J. CellBiol. In press 122. Aderem,A. A., Scott, W.A., Cohn,Z. A. 1986.Evidencefor sequentialsignals in the inductionof the arachidonicacid cascade in macrophages.J. Exp. Med. 163:139-54 123. Pfefferkorn, L. C. 1984. Transmembrane signaling: an ion-flux-independentmodelfor signal transduction by complexed Fc receptors. J. CellBiol. 99:2231-40 124. Suzuki,T., Sadasivan,R., Saito-Taki, T., Stechschulte,D. J., Balentine,L., Helmkamp, G. M. Jr. 1980. Studies of Fcr Receptors of human B lymphocytes: PhospholipaseA2activity of Fcy receptors. Biochemistry19: 6037125. Nitta, T., Suzuki,T. 1982.Biochemical signals transmitted by F%receptors: Triggeringmechanisms of the increased

Annual Reviews

Annu. Rev. Immunol. 1988.6:251-281. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

280

UNKELESS, SCIGLIANO & FREEDMAN

synthesis of adenosine-Y,5’-cyclic monophosphate mediated by F%2a-and Fc 2b-receptors of a murine macrophage-hkecell hne (P388D1).J. ImmunoL 129:2708-14 126. Nitta, T., Saito-Taki, T., Suzuki, T. 1984. Phospholipase A2activity of FC~,2b receptorsof thioglycollate-elicited murine peritoneal macrophages. J. LeukocyteBiol. 36:493-504 127. Fernandez-Botran, R., Suzuki, T. 1985. Biochemicalproperties of phosphatidylcholine-bindingproteins that share common antigenic determinants with the Fc..2b receptor. Biochemistry 24:1896-190’~ 128. Fernandez-Botran, R., Suzuki, T. 1986. Biochemicalsignal transmitted by Fc receptor for immunoglobulin G2aof a murine macrophage-likecell line, P388Dl:modeof activation of adenylate cyclase mediated by immunoglobulinG2abinding proteins. Biochemistry 25:4388-97 129. Green,S. A., Plutner, H., Mellman,I. 1985. Biosynthesis and intracellular transport of the mousemacrophageFc receptor. J. Biol. Chem.260:9867-74 130. Fridman, W. H., Gelabert, M. J., Daeron, M., Moncuit, J., Lowy,I., Theze, J., Neauport-Sautes,C. 1985. IgG-bindingfactors. MethodsEnzymol. 116:403-16 131. Fridman, W.H., Golstein, P. 1974. Immunoglobulin-bindingfactor present on and produced by thymus-processed lymphocytes(T cells). Cell. Immunol.11: 442-55 132. Gisler, R. H., Fridman, W.H. 1975. Suppressionof in vitro antibody synthesis by immunoglobulin-binding factor. J. Exp. Med.142:507-17 133. Neauport-Sautes, C., Fridman, W.H. 1977. Characterization of suppressive immunoglobulin-binding factor 0BF). II. Purification and molecularweight determination of IBF produced by L5178-Ytheta-positive lymphoma.J. Immunol. 119:1269-74 134. Neauport-Sautes, C., RabourdinCombe, C., Fridman, W. H. 1979. T-cell hybrids bear Fc gammareceptors and secrete suppressor immunoglobulinbindingfactor. Nature277: 656-59 135. Lowy,I., Brezin, C., Neauport-Sautes, C., Theze, J., Fridman, W.H. 1983. Isotype regulation of antibody production: T-cell hybrids can be selectively induced to produce IgG1 and IgG2subclass-specific suppressiveimmunoglobulin-bindingfactors. Proc. NatL Acad. Sci. USA80:2323-27

136. Loube,S. R., Dorrington,K. J. 1980. Isolation of biosynthetically labeled Fc-binding proteins from detergent lysates and spent culture fluid of a macrophage-like cell line (P388DI). ImmunoL125:970-75 137. Pure, E., Durie, C. J., Summerill,C. K., Unkeless,J. C. 1984.Identification of soluble Fc Receptorsin mouseserum and the conditioned medium of stimulated B cells. J. Exp. Med.160: 183649 138. Khayat, D., Dux, Z., Anavi, R., Shlomo,Y., Witz, I. P., Ran,M. 1984. Circulating cellfree Fc gamma2b/ gammaI receptor in normal mouse serum:its detectionandspecificity. J. Immunol. 132:2496-2501 139. Khayat, D., Serban, D., Dux, Z., Schlomo, Y., Jacquillat, C. 1986. Roleof infection in the modulationof mousecirculating soluble cell-free Fc gamma 2b/gamma 1 receptor. Scand. J. ImmunoL 24:83-91 140. Neauport-Sautes, C., Dacron, M., Teillaud, J. L., Blank, U., Fridman, W.H. 1986. Theoccurrence, structural and functional properties of immunoglobulinFc receptors on murineneoplastic cells. Intern. Rev. Immunol.1: 237-71 141. Fridman,W.H., Teillaud, J. L., Amigorena, S., Daeron, M., Blank, U., Neauport-Sautes,C. 1987. Theisotypic circuit: immunoglobulins, Fc receptors and immunoglobulinbinding factors. Intern. Rev. Immunol.2:221~t0 142. Blank, U., Fridman, W. H., Daeron, M., Galinha, A., Moncult, J., Neauport-Sautes, C. 1986. Size and charge heterogeneity of murine IgG-binding factors [IgG-BF]. J. Imrnunol. 136: 2975-82 143. Dacron, M., Neauport-Sautes, C., Blank, U., Fridman, W. H. 1986. 2.4G2, a monoclonal antibody to macrophage Fc gammareceptors, reacts with murine T cell Fc gamma receptors and16:1545-50 IgG-bindingfactors. Eur. J. Immunol. 144. Amigorena,S., Moncuit,J., Fridman, W.H., Teillaud, J. L. 1987.Asensitive methodfor testing the effect of immunoglobulin binding factor on Ig secretion by hybridomaB cells. J. ImmunoLMethods 99:57~4 145. Teillaud, J. L., Mathiot, C., Amigorena, S., Brunati,S., Moncuit,J.~ Fridman, W.H. 1987. Regulation of hybridomaB cell proliferation by immunoglobulin binding factor (IBF). CancerDetection and Prevention, New York: Liss

Annual Reviews

Annu. Rev. Immunol. 1988.6:251-281. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

FC~, RECEPTORS 281 146. Calvo, C. F., Watanabe,S., Metivier, uble circulating Fc~receptorsin human D., Senik, A. E. 1986. Humanmonoserum: A newELISAassay for specific cyte cell line (U937)releases supand quantitative detection. ~. Immunol. pressive Methods 100:23~41 Immunol.IgG-binding 16:25-30 factor(s). Eur. J. 150. Hogarth, P. M., Hibbs, M. L., Bona147. Sandor, M., Erdei, A., Blank, U., donna, L., Scott, B. M., Witort, E., Neauport-Sautes,C., Fridman,W.H., Pietersz, G. A., McKenzie,I. F. C. Gergely, J. 1986. Therole of carbo1987. Themurine Fc receptor for IgG hydrates in the IgGbinding and sup(Ly 17): Molecularcloning and specipressive activities of shed humanFc ficity. Immunogenetics. In press receptors. Ann. Inst. PasteurImmunoL 151. Kinet, J. P., Metzger, H., Hakimi, 137D: 79-91 J., Kochan, J. 1987. A ~DNApre148. MacLean,C. A., Goudie, B. M., Macsumptivelycodingfor the ¯ subunit of the receptor with high affinity for Sween,R. N., Sandilands,G. P. 1984. Serum Fc gamma-receptor-like molimmtmog~obulin E. Biochemistry. In ecules in primary biliary cirrhosis: press a possible immunoregulatorymecha- 152. Metzger,H., Alcaraz, G., Hohman, R., nism. Immunology53:315-24 Kinet, J. P., Pribluda, V., Quarto, R. 149. Khayat,D., Geffrier, C., Yoon,S., Sci1986. Thereceptor with high affinity gliano, E., Soubrane, C., Weil, M., for immunoglobulinE. Ann. Rev. ImUnkeless,J. C., Jacquillat,C. 1987.Solmunol. 4:41%70

Annu. Rev. Immunol. 1988.6:251-281. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Annu. Rev. Immunol. 1988.6:251-281. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Annual Reviews www.annualreviews.org/aronline

Annu. Rev. Immunol. 1988.6:283-308. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Ann. Rev. Immunol.1988.6: 283-308 Copyright©1988by AnnualReviewsInc. All rights reserved

MELANOMAANTIGENS: IMMUNOLOGICAL AND BIOLOGICAL CHARACTERIZATION AND CLINICAL SIGNIFICANCE M. Herlyn

and H. Koprowski

The Wistar Institute of Anatomyand Biology, Philadelphia, Pennsylvania 19104 INTRODUCTION Humancutaneous malignant melanoma is, both clinically and experimentally, one of the best studied solid humantumors. Clinical observations on tumor progression have been related to sequential histopathological studies of lesions at all stages of tumor progression. Clark and his collaborators (1) have identified six different steps of tumor progression, starting with the commonacquired melanocytic nevus which does not show any architectural or cytologic atypia. A melanocyte nevus with persistent architectural but no cytological atypia represents step two of the progression. The dysplastic nevus (step three) has features of architectural and cytologic atypia. Although the melanocyte nevus and the dysplastic nevus can be regarded as precursor lesions of melanoma,they usually undergodifferentiation and disappear. Step four of progression is represented by the radial growth phase (RGP) primary melanoma, which shows a strong tendency for invasion of neighboring tissue but does not have demonstrable competencefor metastasis (2). Surgical excision of this lesion, therefore, will lead to cure of the disease. Focal lesions may, however, arise in the RGPlesion to form ultimately the vertical growth phase (VGP) primary melanoma(step five) which has acquired competence 283 0732-0582/88/0410-0283502.00

Annual Reviews

Annu. Rev. Immunol. 1988.6:283-308. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

284

HERLYN & KOPROWSKI

for metastasis.Theincidenceof metastasisis related in linear fashionto the thickness of VGP(3). Surgical excision of VGPlesions requires extensive follow-up of the patients to monitor for the possible developmentof metastasesthat wouldrepresent the sixth step of tumorprogression. Cutaneous malignant melanomaaccounts for 1% of cancer in the UnitedStates, 3%of all skin cancer, but 65%of all skin cancer deaths. Thehighly fatal nature of disseminateddisease is due to the overall poor response of melanoma patients to conventionalchemotherapyor radiation therapy. Recent progress in characterization of antigens expressed and shed by melanoma cells, with monoclonalantibodies (MAbs)used as tools, has helped to develop newapproaches in the diagnosis and therapy of melanoma.Diagnosis of melanomawith MAbswill focus on localization of metastases and the extent of disseminationof the malignancy.Therapeutic strategies involvingthe use of biological responsemodifierssuchas MAbswill aim at the elimination of micrometastasesand prevention of further dissemination.However,clinical responseshavealso beenachieved with MAbtreatment in patients with large metastatic tumor burden, indicating that targeting of melanoma-associated antigens (MAA) may an importantalternative to conventionaltherapy. IMMUNOLOGICAL AND BIOLOGICAL CHARACTERIZATION OF MELANOMAASSOCIATED ANTIGENS The use of murine MAbsagainst MAA,first described by Koprowskiet al (4), led to the subsequentcharacterization of several major antigenic systems,each consisting ofimmunologically and biologically distinct antigenicstructures anda variety of other less well-definedantigens. Figure1 illustrates the expression and/or shedding of MAA representing each system. Extracellular matrix proteins such as fibronectin are secreted in large quantities by melanoma cells but are not expressedon the cell surface. Gangliosidesand high-molecular-weightoncofetal proteins are expressed on the cell surface and are shed; both interact with the substrate, ~ e.g. extracellular matrixproteins. Cell surface receptors for growthfactors and intracellular and extracellular cation transport and binding proteins are involvedwith the intracellular transport of biologically active material. Histocompatibilityantigens that are both expressedand shed by melanoma cells play a role in immunerecognition. Cytoplasmicantigens related to the pigrnented phenotypeof melanocytescan also be found in the spent mediumof pigmentedcells. Sheddingof tumor-associated antigens has recently beenreviewed(5).

Annual Reviews

Annu. Rev. Immunol. 1988.6:283-308. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

MELANOMA=ASSOCIATED ANTIGENS

~

285

Gangliosides ~ I ExtracellularMatrixProteinsI

Figure 1 Schematicillustration of the expression and shedding of melanoma-associated antigens. Pigmentationantigens and extracellular matrix antigens are not expressedon the cell surface; growthfactor receptors and the cation transport and binding proteins are expressed but not shed or minimallyshed. The other antigens, e.g. gangliosides, highmolecular-weight(HMW) oncofetal proteins, and HLAantigen are both expressed and shed.

Cell Substrate Interacting Oncofetal Proteins MAA expressed by fetal cells of different embryologicalorigin mayrepresent the class of oncofetalproteins. In culture, only neural crest-derived cells such as melanomas and gliomasexpress these high-molecular-weight antigens.Thereis little heterogeneity in the expressionof oncofetalproteins betweendifferent melanomasand within each tumor whentheir presence is tested either in tumorsections or on cultured cells. Anothercommon characteristic of oncofetal proteins is their apparent involvementin adhesion,motility, andcell-cell contacts. At least four, andpossiblymore, high-molecular-weight antigens belongto this class. CHONDROITIN SULFATEPROTEOGLYCAN (gp250/>400 kd) Chondroitin

sulfate

proteoglycan (CSP), its encoding gene mappedto chromosome 15 (6), a major MAA that has frequently induced corresponding MAbsbecause of its strong immunogenicity in mice(7, 8 for review). For production MAbs,micewereinjected with either melanoma cells (4, 9-12), nevuscells (M. Herlyn, unpublishedobservations), or gliomacells (13). In more

Annual Reviews

Annu. Rev. Immunol. 1988.6:283-308. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

286

HERLYN & KOPROWSKI

90%of melanomacultures, 80 to 100%of cells are expressing chondroitin sulfate proteoglycan (14-16). Most melanoma cells express between 100,000 and 6,000,000 binding sites (17-19). The chondroitin sulfate proteoglycan is expressed on the melanomacell surface on microspikes which are present as 1-2 # structures on the upper cell surface and as structures up to 20/~ at the cell periphery (20). Peripheral chondroitin sulfate proteoglycan microspikes are involved in the initial interactions between adjacent cells and they form complex footpads that make contact with the substratum. Expression of chondroitin sulfate proteoglycan could be induced in human tumor-mouse hybrid cells when cells were cultured on extracellular matrix instead of on plastic, indicating that cell-matrix interactions provide controlling signals for expression (6). The antigen is abundantly present in adhesion plaques that are deposited along cell membranes.MAbsto chondroitin sulfate proteoglycan do not significantly affect adhesion of melanomacells but block chemotactic and chemokinetic motility of the cells (21) and reduce their colony formation in soft agar (22). Several distinct epitopes have been detected with different MAbson chondroitin sulfate proteoglycan with two (20, 23, 24), three (18, 19), five (6) determinants defined. Antigenic determinants are located either the 250 kd core glycoprotein or on the >400 kd chondroitin sulfate proteoglycan (10, 25), with the heterogeneity largely due to the glycosylation of the molecule (19). 3H-leucine labeling of melanomacells and digestion of the lysate with chondroitinase ABCshowed only the 250 kd component, whereas lysates of melanomacells labeled with 35SO4-2revealed only a component of > 400 kd. The identity of O-linked glycosaminoglycans associated with the chondroitin sulfate proteoglycan was established by alkaline borohydride treatment of 3~S04-2-1abeled immunoprecipitates and subsequent cellulose acetate electrophoresis (8 for review). The MAb9.2.27 recognizes several N-linked glycosylated components at 210 kd and 220 kd which may be either degradative components of the core structure or early glycosylated precursors of the 250-kd core protein (25). The glycosaminoglycan chains released by alkaline borohydrate treatment of the proteoglycan were approximately 60 kd. Based on these studies, it was estimated that the core protein has three attached chondroitin sulfate chains (20). The clinical significance of chondroitin sulfate proteoglycan is discussed below. MELANOMA-ASSOCIATED CELLULAR ADHESION MOLECULE (p130/105 kd) Three, possibly four, MAbshave been described that under nonreducing conditions immunoprecipitate from melanomacells a 130-150 kd and a 90105 kd protein species, and under reducing conditions, 116-120, 95, 29,

Annual Reviews

Annu. Rev. Immunol. 1988.6:283-308. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

MELANOMA-ASSOCIATED ANTIGENS

287

and 25-26 kd species. MAbNu4B(4, 26) binds to all cultured melanomas and gliomas (14) and to most melanomasin situ (27); MAbDA3(16, has a similar binding pattern but higher binding affinity (M. Herlyn, unpublished observations). MAbsAMF7 (21) and DA3 both inhibit attachment of melanomacells to different substrates. The AMF7-antigen, which is abundantly present in adhesion plaques of melanomacells (21), has been designated melanoma-associatedcellular adhesion molecule-1 (MECAM-l) by deVries and coworkers since it plays a role in motility and migration of melanomacells. It is unclear whether the 150/95 kd protein detected by MAb140.72 also belongs to this group because this MAbalso immunoprecipitates a 200 kd glycoprotein corresponding to the CEAmolecule (29). More extensive comparative studies of all four antibodies to confirm their relatedness should be undertaken since they are potentially important in studyi,]g invasion and metastasis of melanomacells. kd) MAb77, obtained by immunizing mice with crude placental membranes, immunoprecipitated from melanomacells a 120/94 kd protein under nonreducing conditions (28). The antibody showsa high degree of specificity for melanomacells in vitro (18) and in situ (D. E. Elder, R. Stuart, M. Herlyn, in preparation). Preliminary results from our laboratory indicate that MAb77 efficiently blocks attachment of melanomacells to substrate and is cytotoxic on melanomacells in the absence of either complementor effector cells within a few hours of incubation with the cells. PLACENTAL MEMBRANEANTIGEN (p120/94

HIGH-MOLECULAR-WEIGHTPROTEINS WITH GANGLIOSIDE-LIKE DISTRIBUTION

PATTERN (p260 kd) Immunization of mice with human melanoma cells followed by booster injections with erythrocytes that were precoated with semipurified gangliosides (28) resulted in several MAbsthat immunoprecipitated from melanomacells proteins of approximately 260 kd. These MAbsshowed remarkable specificity for melanomain vitro and in situ. Antibodies of this group inhibited adhesion of melanomacells to substrate by more than 60% (U. Rodeck, R. Kath, M. Hedyn, unpublished observations). Gangliosides MAbsproduced in several laboratories have defined at least four melanoma-associated gangliosides: GD2(30-32); alkali-labile 9-0-acetylated GD3(33-35); GD3(3 l, 36-39); and GM2(40). Although these antigens are minor brain gangliosides, they are otherwise remarkably restricted in their expression to tumors of neural crest origin as has been shownmost extensively with GD3(41, 42). MAbsto GD2and 9-0-acetylated GD3fail

Annual Reviews

Annu. Rev. Immunol. 1988.6:283-308. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

]288

HERLYN & KOPROWSK1

to bindto intact, adherentcells, whereasthey bindeffectively to cells mechanically or enzymatically dissociatedfromsubstrate(Figure2). Gangliosides are also readilyshedby melanoma cells (32, 35). Like the oncofetal proteins, melanoma-associated gangliosides have beenimplicatedin cell adhesion.It is unclearwhethergangliosidesfunction as receptorsfor fibronectin(43, 44) or play a role in the electrostatic requirementsfor cell-substrate interactions (31). MAbs to GD2andGD3 canblockattachment of cells to the substrate(31, 45, 46), andthe effect of different MAbs to GD2andGD3is additive in blockingattachmentof melanoma cells in vitro (31). Furthermore,anti-GD2/GD3 ganglioside MAb can preventinvasion of melanoma cells in vitro andmetastasis in vivo(46). Melanoma-associated gangliosides interact morefrequentlythan other MAb-definedMAA with the host immunesystem. Humanantibodies

I

4000

80

~ 20

l

2000 o

~

lOOO

~

WM75 WM373 Patient1

WM115 WM266-4 Patient2

Fi#ure 2 Expression and shedding of GD2/GD3 gangliosides as assayed by MAbME36. l on primary (WM75, WM115) and metastatic (WM373, WM266-4) melanomacell lines of two patients. Binding of antibodies was tested on live adherent cells in mixedhemadsorption assays (left bar in each group), live cells in suspension by cytometry (medium bars), and on spent mediumof cells cultured for 5 days in serum-free mediumby solid phase radioimmunoassays (right bars). Thescale on the left vertical axis indicates the percentage of reactive cells in rosetting assays and in indirect immunofluorescence,whereasthe right vertical axis gives cpmin indirect radioimmunoassays.

Annual Reviews

Annu. Rev. Immunol. 1988.6:283-308. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

MELANOMA-ASSOCIATED ANTIGENS

289

to GD2have been produced by cultured lymphocytes obtained from melanoma patients (30, 47) or isolated fromsera of tumor-bearingpatients (48). After immunizationswith melanoma cells, patients developedantibodies to GM2and GD2but not to GD3or GM3(49). Melanomaassociated gangliosides are possibly involvedin activation of suppressor cells (A. Cochran, personal communication),and blocking of GD3with MAb can activate tumor-specific cytotoxic lymphocytes(50). Gangliosides play a potentially important role in growthregulation and differentiation of melanoma cells, similar to the stimulatoryeffect of gangliosideson astroglial cell proliferation (51) or the inductionof monocyte differentiation in promyelocyticleukemiacells (52). Moreextensive studies of this role of gangliosidesare in progress. Receptors for Growth Factors Thereare at least six growthfactor-growthfactor receptor systemsactive in humanmelanoma cells: (a) epidermalgrowthfactor (EGF)/alphatransforming growth factor (TGF); (b) nerve growthfactor (NGF);(c) lin/insulin-like growthfactor; (d) platelet-derived growthfactor (PDGF); (e) beta-TGF;and (f) fibroblast-growthfactor (7 for review). In culture, the EGFreceptor is expressed on advanced primary and on metastatic melanoma cells most prominentlyif cells contain an extra copyof chromosome 7 whichencodes the gene for this receptor (53). In situ, it strongly expressed on advancedmetastatic cells. EGFis mitogenic for metastatic melanoma cells whencultured in chemically defined medium, evenwhenthe number of bindingsites is belowthe detection level (54, 55). The NGFreceptor, encoded on chromosome 17 (56), is expressed nearly all cultured melanoma cells, with approximately 50,000to 2,000,000 binding sites per cell (see 57 for review). A 75 kd glycoprotein wasimmunoprecipitatedwith MAbsfromexternally labeled melanoma cells (58). Nodata are available on whetherNGFhas any growthmodulatoryeffects on melanoma cells. Thepotential role of other growthfactor/growthfactor receptor systemson melanoma cells has recently been discussed(7). Cation Transport and Binding Proteins MELANOTRANSFERRIN (p97 kd) Like chondroitin sulfate proteoglycan, p97 kd is very immunogenicin mice whenmelanomacells are injected for the production of MAbs.Brown& Hellstrom and their associates have extensivelystudied the tissue distribution of p97 kd (59-61). In culture, this protein is highlyexpressedby almostall melanoma cells as well as by cells of a few carcinomas.In situ, most melanomas bind MAbsto p97 kd. In fetal tissues, p97kd is foundmostlyin the colonic mucosaandin adult tissues on sweatglandcells.

Annual Reviews

Annu. Rev. Immunol. 1988.6:283-308. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

290

HERLYN & KOPROWSKI

Thereare at least five different antigenic determinantson p97 kd (62). The protein is a monomeric sialoglycoprotein with intrachain disulfide bonds. N-terminal amino acid sequencing revealed homologyin 7 of 12 residues with transferrin andlactotransferrin (63), and since purified p97 can bind iron, it has a functionalrelationship to transferrin. Purified and cloned p97 mRNA encodes a 738-residue precursor, and the mature p97 moleculecomprises extracellular domainsof 342 and 352 residues and a C-terminal 25-residue stretch of predominantlyuncharged and hydrophobicaminoacids, whichmayact as a membrane anchor (64). Eachextracellular domaincontains 14 cysteine residues, whichform 7 intradomaindisulfide bridges, and possiblyoneor twoglycosylationsites. Theconservationof disulfide bridges and aminoacids thought to compose the iron bindingpocketssuggest that p97 is also related to transferrin in tertiary structure and function. This has led Roseand coworkers(1986) to suggest for p97 the name"melanotransferrin." CALCIUM-BINDING S-100S- 100 protein is a highly acidic cytoplasmicprotein with a molecularweightof 21 kd whenisolated frombovinebrain extracts (65). It is composed of two peptide chains whichassociate as dimers(66). TheS-100protein is part of a family of calciumbinding proteins, which VanEldik(67) called calcium-modulatingproteins and whichinclude calmodulin.Althoughthe S-100protein is highly characteristic for neural crest-derivedtumors,includingmelanoma cell lines (68), it is also detected in other normaland malignanttissues (69). Antibodiesto S-100are widely used for the immunohistologicaldiagnosis of nonpigmentedmelanomas (69). Class H HLA Antigens HLAclass II antigens comprise the gene products of the DR,DQ, and DP loci located in the HLA-Dregion of chromosome6. HLA-DR is strongly expressed on cells of approximately 75%of cultured melanoma lines (70, 71, see 72 for review).Expressionof all three types of HLA class II antigens in melanoma metastasesdoes not correlate with: (a) anatomic sites of the lesions; (b) expressionandcellular distribution of HLA class antigens; and/or (c) expression of several different MAA (73). mentation and tumorigenicity in nude mice are also unrelated to HLADRexpression (74). Of the metastatic lesions, approximately50%(75) express HLA-DR, with considerable heterogeneity in the percentage of positive cells in lesions obtainedfromthe samepatient or fromdifferent patients (76). In primary melanomas,39%of lesions showpresence antigen in more than 10%of cells (77), and the occurrence of HLA-DRpositive tumorcells indicates in such lesions usually a high metastatic potential of primarytumors, independentof tumorthickness (78).

Annual Reviews MELANOMA-ASSOCIATED ANTIGENS

291

Annu. Rev. Immunol. 1988.6:283-308. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

HLA-DR on primary melanoma cells appears to be involved in the cellmediated immuneresponse. Tumorcell lines derived from early lesions and whichexpress HLAclass II antigens stimulate autologous T-cells to undergoblastogenesis whencocultured in vitro (79-81). There is quantitative relationship between the expression of HLA-DR by tumor cells and the degree of lymphocyteproliferation. Onthe other hand, cell lines from advancedlesions are not stimulatory for autologousT-cells, regardless of whether they express HLA-DR. Pigmentation-Associated

Antigens

Three antigens have been characterized with MAbsthat are associated with pigmentation both in normal and malignant melanocytes. A 70-80 kd antigen is foundin the melanosomes of pigmentedcells but is apparently not associated with tyrosinase (82). Other MAbsdefine 50/18/17kd proteins that are only present in pigmentedcells (83, 84), whereasMAb HMSA-1 binds not only to melanosomesbut also to the endoplasmic reticulum of nonpigrnentedmelanoma cells (85). Highly Glycosylated

Neuroglandular Antigen

A highly glycosylated protein with a molecular weightof 30~0kd and a protein core of 20 kd has been identified by anti-melanomaMAbsin several laboratories(86-89),but it is yet unclassifiedin relation to other antigens describedin Figure 1. Theheterogeneityin chargeand molecular weight of this antigen result from non-uniformprocessing of a single protein core whose amino acid sequence does not correspond to any hitherto described protein (90). The antigen is an excellent immunohistological markerin fixed paraffin-embedded tissue sections. It is not only widely expressed on melanomasbut also on carcinomas(91). Based on the expression on normalhumantissues, Sikora et al (89) termedthe 30-60 kd antigen "neuroglandularantigen." Extracellular Matrix Proteins Extracellular matrixproteins are secreted in large quantities by melanoma cells and they maybe of importancein adhesion, motility, and invasion. Lamininandcollagen type IV havespecific distribution patterns in malignant melanocytelesions whichcan be used as diagnostic markers (92). Fibronectin is also secreted by cultured melanoma cells (7), and wehave recently producedMAbsagainst a 230 kd protein(s) that is specifically secreted by tumor cells of neural crest origin, including melanoma (M. Herlyn, unpublishedobservations).

Annual Reviews

292

HERLYN & KOPROWSKI

Annu. Rev. Immunol. 1988.6:283-308. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Differentiation

Antigens

Several antigens on melanoma cells are associated with differentiation of melanomacells, such as the nevus antigen described by Nakanishi & Hashimoto(93). Since melanocytesare of neural crest origin, neuronal antigens such as the MAbA2B5-definedganglioside antigen, galactocerebroside(J. Jambrosic,personal communication), or myelin-associated glycoprotein (94) are found on melanoma cells. The MAbreacting with myelin-associated glycoprotein (100 kd) was originally producedafter immunizationof mice with humanlymphoblastoidcells. The MAbreacts with a carbohydrate determinant shared by different protein antigens expressedby natural killer cells and cells of the peripheral nervoussystem (95). Houghton et al (96) and Real et al (41) haveproposeda differentiation pathwayin melanomabased on morphological and antigenic studies of cultured melanocytesand metastatic melanoma cells. Thoseauthors distinguish three differentiation stages: early, intermediate,andlate. Early, "poorly differentiated" melanomacells express EGFreceptor, HLA-DR, proteoglycan, and A010antigen. Late, "highly differentiated" melanoma cells expressmarkersassociated with pigmentation,e.g. p75. Intermediate cells expressgp110and Calla. ANTIGENS DEFINING CELLS AT VARIOUS STAGES OF TUMOR PROGRESSION IN THE MELANOCYTE SYSTEM Antigen Expression on Cultured Cells Considerable differences in antigen expression are observed between normal, precursor, and malignantmelanocytesmaintainedeither in vitro or in situ. As shownin Figure 3, cultured normalmelanocytesexpress most melanoma-associatedantigens, with the exception of GD2and HLADR(87). The expression of tumor-associatedantigens on cultured normal melanocytesbut not on melanocytesin situ could indicate that the melanocytes grownin culture have acquired antigenic properties of transformedcells. However,observing more than 500 cultured specimens, we havenot seen any spontaneoustransformationof the cells. Normalmelanocytes have always had a diploid karyotype, do not grow in soft agar, have a finite life span, and do not form tumors in nude mice (see for review). This expression of tumor-associated antigens on cultured melanocytesbut not on cells in situ could be due to differences in growth rate since melanocytesin culture rapidly proliferate whereasmelanocytes in situ do not. HLA-DR can be induced in normal melanocytesby gamma-

Annual Reviews ANTIGENS MELANOMA-ASSOCIATED In vitro

293

In situ

p120(B) p~~O/105

Annu. Rev. Immunol. 1988.6:283-308. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

GDa

p120/94 Figure 3 Expression of cell surface antigens on normal humanmelanocyteswhenmaintained in vitro or whentested in sectionsof normalskin. For further descriptionof antigens see section on Melanoma-Associated Antigens.p120(A) and (B) refer to different antigens with samemolecularweight. interferon as has been demonstrated in several laboratories (97, 98, see 72 for review). HLA-DRand GD2 expression on melanocytes can also be induced or enhanced by transformation of cells with murine sarcoma viruses (99). Cultured melanocytes and nevus cells express two antigens, p 145 kd and p120 kd, which are not detected on advanced primary or metastatic melanoma cells (Table 1). Melanocytes of newborn foreskin express higher Table 1 Expression of melanoma-associatedantigens on cultured melanocytesisolated from various a’~ stages of tumorprogression Primary melanoma cAntigen p145 kd p120 kd HLA-DR GD2/GD 3 Proteoglyean NGFreceptor

Normal Antibody skin 487 207 13-17 ME36.1 ME31.3 ME20.4

+d + ++ ++

Nevus RGP + + + + + + +

_ + + + + + +

Metastatic VGPmelanoma References

+ + + +

_ + + + +

+ + + +

+ + + +

_ + + + +

+ + + +

28 16 4, 16 32 16, 23 16, 58

a All assaysweredoneonlive adherentcells exceptfor MAb ME36.1whichwastestedoncells in suspension. bat least 20specimens weretestedfromeachcell typeexceptthat only5 RGP cell lines weretested. ~ References referto the initial production of monoclonal antibodies andto the characterization of binding pattern. d Expression:+ + + = strong; + + = moderate;+ = weak;- = none.

Annual Reviews 294

HERLYN & KOPROWSKI

Annu. Rev. Immunol. 1988.6:283-308. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

concentration of antigens than those obtained fromadult skin (11). HLADRantigen is first expressed on nevus cells and at highest levels on metastatic melanoma cells (15, 16, 100). GD2expressionreflects the malignant phenotypeof melanocytesmorethan any other antigen defined with MAbs (39). Thetotal gangli0sideamountas well as the distribution of each ganglioside mayvary. Cultured melanoma cells, in contrast to biopsied material, express GD2and GM2more strongly than other gangliosides (101).

ANTIGEN EXPRESSION ON CELLS IN SITU Onmelanocytesin situ, only GD3of all the MAA is expressed, but only at low levels (41, 42). MostMAbs describedbefore bind in situ to cells nevi and melanoma, althoughquantitative differences in binding mayexist betweenthe different lesions (Table 2). Highestexpressionof antigens generally found on metastatic cells. Decreasedexpression on the most advanced melanomascan be seen for proteoglycan (102) and p30-60 antigen(86). Selective screening of MAbson melanoma sections has defined several newantigens that werenot previously found on cultured cells. Holzmann et al (102) recently proposeda conceptof tumorprogressionbased on the antigenic phenotypeof cells in situ using mostlyMAbs that wereselected on tissue sections. Eachcell type expressedcharacteristic antigens. Dysplastic nevuscells expressin addition to proteoglycan,the gpl30 antigen (103) and p76 antigen (104); early primarymelanoma cells in addition the other antigens express gp75 (105) and the PAL-M1 (106) antigens; Table 2 Expression in situ of antigens on melanocytes of various stages of tumor proagression Primary melanoma Antigen

Antibody

EGFreceptor GD2 HLA-DR NGFreceptor Proteoglycan p30-60 GD3

425 ME36.1 13-17 ME20.4 ME31.3 ME49.1 ME 24

Normal skin _b +

Nevus

RGP

-_ ___ + + + _ +

-___ _ + + + + + + + +

VGP

Metastatic melanoma

+

+ + +

+ + + + + + + + + + + + +

+ + + + + + + + + + + + + +

a All immunoperoxidase assays weredone on frozen sections by D. E. Elder exceptfor ME49.1 which wastested on fixed tissue sections(86). bExpression: + + + + = very strong; + + + = strong; + + = moderate; + = weak; - = none.

Annual Reviews MELANOMA-ASSOCIATED ANTIGENS

295

Annu. Rev. Immunol. 1988.6:283-308. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

advanced primary melanoma cells express in addition gp89 (107) and gp 113 (104) antigens. Such antibody panels could prove useful in defining in situ the different stages of tumor progression. A disadvantage of this panel is that the antigens defined are sensitive to fixation and dehydration procedures. All immunohistological studies, therefore, must be performed on frozen tissues which often lack the structural integrity found in fixed tissues. An alternative that would allow retrospective studies might be the selection of MAbsin fixed tissue sections (108). However,the latter approach has not yielded manyMAbswith restricted binding patterns.

MELANOMA ANTIGENS AS TARGETS FOR IMMUNODIAGNOSIS ImmuneHistology and Serodiagnosis with Monoclonal Antibodies The immunohistological diagnosis of melanoma with MAbsdefining MAA has not been applied widely in the histopathology laboratory mainly because most antigens are sensitive to fixation and dehydration procedures. The acidic S-100 protein and the highly glycosylated p30-60 kd antigen are both stable after fixation. Preliminary results from our laboratory indicate that the p260antigens, which have specificities similar to gangliosides, are, unlike the gangliosides, stable after fixation. The serodiagnosis of melanomais possible with MAbsdetecting shed antigens in melanomapatients’ sera (see 5 for review). Five antigens have been detected in sera of patients with advanced metastatic melanoma: HLA-DR (109), proteoglycan (110-112), p30-60 kd (113), pl00 kd and GD2(unpublished). GD2has also been found in sera of patients with neuroblastoma (115, 116). Noneof these diagnostic assays has been widely applied in testing large numbersof patients’ sera. In contrast to other cancers, such as gastrointestinal carcinomas, serodiagnosis of melanoma by detecting MAA with MAbsis of minor clinical significance.

Immunodetection with Radiolabeled Monoclonal Antibodies Several MAbshave been used in the immunodiagnosis of melanoma, initially with tumors grown in athymic nude mice and eventually in human patients. For successful tumor localization, several parameters must be optimized. (a) Antibody specificity: MAbsstrongly cross-reactive with normal cells, for example in the bone marrow, will most probably not serve to distinguish the tumor and maycause side effects (117). (b) State of MAbfragmentation: F(ab’)2 fragments of MAbsshow enhanced localization as comparedto intact antibody (118). A practical consideration also the ability routinely to purify and obtain fragments of a particular

Annual Reviews

Annu. Rev. Immunol. 1988.6:283-308. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

296

HERLYN & KOPROWSKI

MAb.For example, purification and fragmentation of IgG3 and IgM antibodies is technically difficult. (c) Radionueleotide:~3~-Iodineand ’~-Indiumhave been mostwidely used in experimentalanimals and in patients (see 119 for review). However,other isotopes maybe moreuseful for imaging,once appropriate coupling techniques have been developed. (d) MAb binding affinity and numberof binding sites per tumorcell. The data in Table 3 demonstratefor two anti-melanomaMAbsthat successful localization of radiolabeled MAbin xenotransplants of humantumors in nude mice dependson a high association constant for a given MAb,such as ME28-8 in contrast to MED63(120). Antibodies with low affinities require a considerablyhigher numberof target sites on the cell surface in order to localize tumorsin vivo. (e) Shedding of tumor-associatedantigens: Potentially, circulating antigen mayabsorbradiolabeled MAbs.In studies with earcinoembryonic antigen and other antigens of gastrointestinal adenocarcinomas,sheddingof tumorantigens into circulation (see 5 for review) did not prevent localization on tumor cells of radiolabeled MAb administered parentally (121,122). These studies indicate that melanomas may be localized with radiolabeled MAbs that detect either cell-boundor shed MAA. Imaging studies in melanomapatients have been done with MAbsto melanotransferrin and to chondroitin sulfate proteoglycan. Larson and collaborators (123) performedthe first studies in patients with anti-p97 MAb.Of lesions larger than 1.5 cm, 85%werevisualized using ~3~-Iodine Table 3 Localization of melanomasin nude mice with radiolabeled monoclonal antibodies ~ correlates with antibody affinity ~25I-Antibodyb Assay system

ME 28-8

ME D63

Scatchard analysis in vitro Bindingsites/cell Percent Association constant 1/mol Percent

5.4 × 106 100% s0.1 x l0 100%

0.31 × 106 5.6 s14 x l0 14,000

Biodistribution in nude mice Localization index¢ (day 4) Percent

0.9 100 %

15.9 1766

a Thesamemelanoma cell line, WM 9, wasused for all studies. b F(ab’)2fragmentswere used for studies in vivo. ME28-8 defines the HLA class II antigens DRand DQ,and MED63,the transferrin related p97kd. ° Localizationindexis calculatedfromthe ratios of specificto non-specificactivity in tumordividedby the sameratio in blood(120).

Annual Reviews

Annu. Rev. Immunol. 1988.6:283-308. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

MELANOMA-ASSOCIATED ANTIGENS

297

for labeling of MAb.Halpern et al (124) and Murrayet al (125) Ill-Indium for localization studies in patients. Imagingof metastatic lesions in both studies wassuccessful in 50%and60%of patients. Unfortunately, accumulatingradiolabeled anti-p97 MAbsat relatively high concentrations in the liver limited the use of anti-p97 MAbsfor imaging studies. MAbsto chondroitin sulfate proteoglycan have been used in patients by Buraggiet al (126, 127) and Siccardi et al (128) whofound specific accumulationof radioactivity in 10 out of 18 and 250 of 412 metastases, respectively. In a systematic kinetic study of l~-Indium-labeledantibody to proteoglycan, Eger and coworkers(129) determinedthat approximately 3.5 mgof antibodyper injection wasrequired for sufficient saturation of the tumorcell surfaces to prevent further binding of MAb.Thesestudies demonstrate that melanomascan be localized by radiolabeled MAbsbut it remainsunclear whetherp97 and chondroitinsulfate proteoglycanantigen are optimaltargets for routine diagnostic studies. Althoughup to now the MAbsreacting with gangliosides are of IgG3 or IgMisotypes and therefore unsuitable for radiolabeling, switchingof the isotypes (32) may allow testing of gangliosideantigens for radiolocalization with MAbs. IMMUNOTHERAPY OF MELANOMA WITH MONOCLONAL ANTIBODIES DEFINING MELANOMA ANTIGENS MAbsto melanomacan suppress growth of tumors in experimental animals and in patients. Inhibition of tumor growth dependson antibody isotype (130). OnlyMAbsof IgG2aand IgG3can inhibit growthof human tumorsxenotransplantedinto nudemice, whereasno inhibition is observed with antibodies of IgG1, IgG2b,IgM,and IgA isotype (Table 4). The ability of IgG2a MAbsto inhibit tumor growth in nude mice strongly correlates with their reactivity in antibody-dependentmacrophage-mediatedcytotoxicity assays (130). IgG2aMAbsthat are reactive in vivoare also reactivein these assaysin vitro (Table5; see 131for review). Humanmonocytesand humanlymphocytesmediate similar cytotoxicity reactions, whichalso dependon the IgG2aisotype of MAbs(132, 133). Complement, however,mediates lysis of target cells with MAbsof IgG3 and IgMisotypes. Suppression of melanomagrowth in nude mice by IgG2a MAbscorrelates with the density of antigenic sites on melanoma cells (17). Tumors with less than approximately300,000antigenic binding sites per cell are not suppressed, whereasthose with morethan one million binding sites are (Table6). Affinityof antibodiesfor target cell antigenapparentlyplays

Annual Reviews

298

HERLYN & KOPROWSKI Table 4 Growth inhibition of humantumors in nude mice with amonoclonal antibodies is dependent on antibody isotype

Annu. Rev. Immunol. 1988.6:283-308. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Isotype

of MAb

IgG1 IgG2a IgG2b IgG3 IgM IgA

Numberof antibodies suppressing btumors/total tested 0/15 9/13 0/2 2/4 0/4 0/1

a Results shownare for melanomas and other humantumors. ~ Inhibition studies weredone by injecting micesubcutaneouslywith 5-10 × 106 humantumor cells and with MAbat approximately200 pg for 5 to 14 days intraperitoneally (130). Growthof tumors wasmonitored weeklyfor durationof the experiments.Positiveinhibition wasconfirmedbystatistical analysis.

a lesser role in therapythan in imagingtrials. SeveralMAAs havebeenused as target antigens for in vitro andin Vivolysis of melanomas. Antibodiesto p97that detect different antigenic bindingsites can synergistically act in complement-dependent cytotoxicity (134). Anti-proteoglycan MAbsdid not lyse tumorsin nudemice(17) unless they werecoupledwith syngeneic effector cells with polyethyleneglycol (135). Among all anti-MAA MAbs, those that detect disialogangliosidesare the strongestmediatorsfor effector cell-mediatedlysis of tumorcells in nudemice(32, 46, 136, 137). Thenew switch variant MAbsof IgG2aisotype (32; Z. Steplewski, M. Thurin, H. Koprowski,unpublishedobservations) will greatly improveavailability of cytotoxic MAbs. Preliminary immunotherapeutic trials have been performedin patients using MAA as target antigens for the cytotoxic effects of MAbs(see 138 for review). Anti-GD3MAbinduced clinical responses in 5 out of 12 melanoma patients (139). Encouragingpreliminary results have also been obtained with an MAbto GD2(D. Guerry, A. Lichtin, D. Herlyn, unpublished observations; J. Y. Douillard, Z. Steplewski, unpublishedobservations), whereas MAbsto p97 and proteoglycan do not appear to be useful for immunotherapyin patients (140, 141). On the other hand, significant intratumor binding of anti-proteoglycan MAbwas found in patients whohad received 200-500mgof MAb(142). CONCLUSIONS Morethan 40 different melanoma antigens have been defined with MAbs. Immunologicaland biological studies have been carried out to assign

Annu. Rev. Immunol. 1988.6:283-308. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Annual Reviews MELANOMA-ASSOCIATED ANTIGENS

299

Annual Reviews 300

HERLYN & KOPROWSKI

Annu. Rev. Immunol. 1988.6:283-308. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Table6 Suppression of melanomagrowth in nude mice by IgG2a monoclonalantibodies acorrelates with antibody(antigen) density on melanoma cells Assay System Numberof mice with suppressed melanomagrowth/total tested Percentagespecific lysis in macrophage-mediated cytotoxicity Percentagepositivecells in indirect immunofluoreseence Number of binding sites per cell Associationconstant (1/mole)

bME37-7

bMEC44

6/6

0/9

77.7

1.8

97.5 78.7 3.6 x 106 0.2 × 106 2.6 x 108 1.5 × 108

"Metastatic melanoma cell line WM 9 wasusedfor all in vitro andin vivostudies(17). bME 37-7detectsthe HLA class II antigensDRandDQ,whereasMEC44definestransferrin-related p97kd.

functions to manyof these antigens. Aprominentfeature of several oncofetal proteins and gangliosidesis their involvementin the adhesionof the melanoma cells to substrate, in cell motility, andin cell-cell interactions. Since these properties are a part of the metastatic cascade, MAbsto such antigens maybe important in the developmentof newtherapeutic approaches. For example,MAbsto melanomagangliosides can effectively inhibit invasion in vitro and tumormetastasis in experimentalanimals. Studies of MAA have helped to define the stages of tumorprogression in situ. In tissue culture, however,most MAAs are already expressed by normalmelanocytes.Thus, either someof the MAAs are expressed by all cells growingin vitro or, alternatively, those cells that can be maintained in tissue culture already express the malignantphenotype. Multicenter comparativestudies with the exchangeof reagents will be required for in depth analyses of the association of antigens with tumorprogression in vitro andin situ. Diagnosis of melanomawith radiolabeled MAbsor by detecting circulating antigens in patients’ sera will probablyremainlimited to studies in specialized centers. Onthe other hand, the preliminarystudies on the therapy of melanomawith anti-ganglioside MAbsare encouraging and should be performed on a muchlarger scale since melanomapatients generally respond poorly to conventional chemo-and radiation-therapy. ACKNOWLEDGMENTS Research summarizedin this review was supported by grants CA-25874, CA-10815,and CA-44877 from the National Institutes of Health and grant IM-402from the AmericanCancer Society. Wegratefully acknowledge the secretarial assistanceof A. Brandeis.

Annual Reviews MELANOMA-ASSOCIATED ANTIGENS

301

Annu. Rev. Immunol. 1988.6:283-308. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Literature Cited 1. Clark, W.H., Elder, D. E., Guerry,D., Epstein, M. E., Greene, M. H., VanHom,M. 1984. A study of tumor progression: the precursor lesions of superficial spreadingandnodularmelanoma. Hum.Pathol. 15:1147-65 2. Elder, D. E., Guerry, D., Epstein, M. N., Zehngebot,C., Lusk, E., VanHorn, M., Clark, W.H. 1984.Invasive malignant melanomaslacking competence for metastasis. Am.J. Dermatopathol. 6 (Suppl. 1): 55-62 3. Clark, W.H., Elder, D. E., VanHorn, M. 1986. Thebiologic formsof malignant melanoma.Hum.Pathol. 17: 44352 4. Koprowski, H., Steplewski,Z., Herlyn, D., Herlyn, M. 1978. Study of antibodies against humanmelanomaproducedby somatic cell hybrids. Proc. NatL Acad. Sci. USA75:3405-9 5. Herlyn, M., Rodeck, U., Koprowski, H. 1987. Sheddingof tumor-associated antigensin vitro andin vivo. Adv.Cancer Res. 49:189-221 6. Rettig, W.J., Real, F. X., Spengler, B. A., Biebler, J. L., Old, L. J. 1986. Humanmelanomaproteoglycan: expression in hybrids controlled by intrinsic andextrinsic signals. Science 231:1281-84 7. Hedyn,M., Clark, W.H., Rodeck,U., Mancianti, M. L., Jambrosic, J., Koprowski,H. 1987. Biologyof tumor progression in humanmelanocytes. Lab. lnvest. 56:461-74 8. Reisfeld, R. A., Cheresh,D. A. 1987. Humantumor antigens. Adv. Immunol. 40:323-78 9. Natali, P. G., Cavaliere, A., Bigotti, M. R., Nicotra, C., Russo,A., Ng, K., Giacomini,P., Ferrone, S. 1983. Antigenic heterogeneityof surgically removedprimary and autologous metastatic humanmelanomalesions. J. Immunol. 130:1462-66 10. Bumol,T. F., Reisfeld, R. A. 1982. Uniqueglycoprotein-proteoglycancomplex defined by monoclonalantibody on humanmelanoma cells. Proc. Natl. Acad. Sci. USA79:1245-49 11. Houghton,A. N., Eisinger, M., Albino, A.P., Cairncross,J. G., Old,L. J. 1982. Surface antigens of melanocytesand melanomas: markers of melanocyte differentiation and melanoma subsets. J. Exp. Med. 156:175~66 12. Hellstrrm, I., Garrigues,J., Cabasco, L., Mosely, G. H., Brown, J. P., Hellstrrm,K. E. 1983.Studies of highmolecular-weight humanmelanoma-

associated antigen. J. Immunol.130: 1467-72 13. Cairncross,J. G., Mattes,M.J., Beresford, H. R., Albino, A. P., Houghton, A. N., Lloyd,K. O., Old, L. J. 1982. Cell surface antigens of humanastrocytomasdefined by mousemonoclonal antibodies: identification of astrocytomasubsets. Proc. NatLAcad.Sci. USA 79:5641-45 14. Herlyn, M., Clark, W. H., Mastrangelo, M.J., Guerry,D., Elder, D. E., LaRossa,D., Hamilton,R., Bondi, E., Tuthill, R., Steplewski,Z., Koprowski, H. 1980. Specific immunoreactivityof monoclonal antimelanomaantibodies to cultured cells and freshly-derived humancells. CancerRes. 40:3602-9 15. Herlyn,M., Steplewski,Z., Herlyn,D., Clark, W.H., Ross, A. H., Blaszczyk, M., Pak, K. Y., Koprowski,H. 1983. Production and characterization of monoclonalantibodies against human malignantmelanoma. Cancerlnvest. 1: 215-24 16. Herlyn, M., Thurin, J., Balaban, G., Bennicelli,J. L., Herlyn,D., Elder, D. E., Bondi, E., Guerry,D., Nowell,P. C., Clark, W.H., Koprowski,H. 1985. Characteristics of cultured human melanocytesisolated from different stages of tumor progression. Cancer Res. 45:5670-76 17. Herlyn, D., Powe, J., Guerry, D., Herlyn, M., Koprowski,H. 1985. Inhibition of human tumor growth by IgG2a monoelonalantibodies correlates with antibody density on tumor ceils. J. lmmunol.134; 1300-4 18. Giacomini,P., Natali, P. G., Ferrone, S. 1985. Analysisof the interactions betweenhumanhigh-molecular-weight melanoma-associated antigens and the monoclonal antibodiesto three distinct antigenic determinants. J. lrnmunol. 135:696-702 19. Ziai, M. R., Imberti, L., Nicotra, M. R., Badaracco,G., Segatto, O., Natali, P. G., Ferrone, S. 1987. Analysiswith monoclonalantibodies of the molecular and cellular heterogeneity of humanhigh-molecular-weight melanoma-associatedantigen. CancerRes. 47:2474-80 20. Garrigues, H. J., Lark, M. W., Lara, S., Hellstrrm, I., Hellstrrm, K. E., Wight, T. N. 1986. The melanoma proteoglycan:restricted expressionof microspikes, a specific microdomain of the cell surface. J. Cell Biol. 103: 1699-1710

Annual Reviews

Annu. Rev. Immunol. 1988.6:283-308. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

302

HERLYN & KOPROWSKI

21. de Vries,J. E., Keizer,G. D., te Velde, Cassidenti, A., Paulson, J. C. 1982. A. A., Voordouw,A., Ruiter, D., Identification of a human neuroRumke,P., Spits, H., Figdor, C. G. ectodermal tumor antigen (OFA-1-2) 1986. Characterization of melanomaas ganglioside GD2.Proc. Natl. Acad. associatedsurface antigensinvolvedin Sci. USA79:7629-33 the adhesion and motility of human 31. Cheresh,D. A., Pierschbacher,M.D., melanoma cells, lnt. J. Cancer38: 465Herzig, M. A., Mugoo,K. 1986. Di73 sialoganglioside GD2and GD3are 22. Harper, J, R., Reisfeld, R. A. 1983. involved in the attachment of human Inhibition of anchorage independent melanomaand neuroblastomacells to growth of humanmelanomacells by a extracellular matrix proteins. J. Cell monoclonalantibody to a chondroitin Biol. 102:688-96 sulfate proteoglycan. J. Natl. Cancer 32. Thurin, J., Thurin, M., Kimoto,Y., Inst. 71:259-64 Lubeck,M. D., Herlyn, M., Elder, D. 23. Ross,A. H., Herlyn, M., Ernst, C. S., E., Smereczynska,M., Karlsson, K.Guerry,D., Bennicelli,J., Ghrist,B. F. A., Clark, W. H., Steplewski, Z., D., Atkinson, B., Koprowski,H. 1984. Koprowski,H. 1987. MonoclonalantiImmunoassayfor melanoma-associbody-definedcorrelations in melanoma ated proteoglycan in the sera of pabetweenlevels of GD2and GD3antitients using monoclonaland polyclonal gens and antibody-mediated cytoantibodies. CancerRes. 44:4642-47 toxicity. CancerRes. 47:1229-33 24. Harper, J. R., Bumol,T. F., Reisfeld, 33. Cheresh,D. A., Reisfeld, R. A., Varki, A. P. 1984. O-acetylationof disialoR. A. 1984. Characterization of monoclonalantibody 155.8 and partial ’ ganglioside GD3by humanmelanocharacterization of its proteoglycan macells creates a unique antigenic antigen on humanmelanomacells. J. determinant. Science 225:844-46 Immunol. 132:2096-2104 34. Cheresh,D. A., Varki,A. P., Varki,N. 25. Bumol,T. F., Walker,L. E., Reisfeld, M., Stallup, W.B., Levine,J., Reisfeld, R. A. 1984.Biosyntheticstudies ofproR. A. 1984. A monoclonal antibody teoglycans in humanmelanomascells recognizesan O-acetylatedsialic acid with a monoclonalantibody to a core in a humanmelanoma-associatedganglycoprotein of chondroitin sulfate glioside. J. Biol. Chem.259:7453-59 proteoglycan. J. Biol. Chem. 259: 35. Thurin,J., Herlyn, M., Hindsgaul,O., 12733-41 Karlsson,K.-A., Stromberg,N., Elder, 26. Mitchell,K. F., Fuhrer,J. P., SteplewD. E., Steplewski, Z., Koprowski,H. ski, Z., Koprowski,H. 1981.Structural 1985. Proton NMR and fast atombomcharacterization of the "melanomabardmentmassspectrometry analysis specific" antigen detected by monoof melanoma-associated ganglioside clonal antibody 691 I 5Nu-4-B.Mol. 9-0-acetyl-GD3. J. Biol. Chem.260: Immunol. 18:207-18 14556-563 27. Thompson, J. J., Herlyn, M., Elder, D. 36. Dippold, W.G., Lloyd, K. O., Li, L. E., Clark, W. H., Steplewski, Z., T. C., Ikeda, H., Oettgen,H. F., Old,L. Koprowski, H. 1982. Use of monoJ. 1980.Cell surface antigensof human clonal antibodiesin detectionof melanmalignantmelanoma:definition of six oma-associated antigens in intact antigenic systems with mousemonohumantumors. Am. J. Pathol. 107: clonal antibodies. Proc. Natl. Acad. 357-61 Sci. USA77:6114-18 28. Herlyn, M., Rodeck, U., Mancianti, 37. Pukel, C. S., Lloyd,K. O., Travassos, M.L., Cardillo, F. M., Lang,A., Ross, L. R., Dippold,W.G., Oettgen,H. F., A. H., Jambrosic, J., Koprowski,H. Old, L. J. 1982. GD3--a prominent 1987. Expressionof melanoma-associganglioside of human melanoma: ated antigensin culturedrapidly dividdetection and characterization by ing humanmelanocytes. Cancer Res. mousemonoclonalantibody. J. Exp. Med. 155:1133-47 47:3057-61 29. Liao, S.-K., Kwang,P. C., Clarke, B. P38. Hellstrrm, I., Brankovan,V., HellJ., Dent,P. B., Ryan,E. D., Khosravi, strrm, K. E. 1985. Strong antitumor activities with IgG3 antibodies to a M.J., Laferte, S., Krantz,M.J. 1985. Monoclonal antibody recognizing humanmelanoma-associatedgangliohuman melanoma-carcinoma crossside. Proc. Natl. Acad. Sci. USA82: reacting oncofetal antigen epitopically 1499-1502 associated with carcinoembryonic anti39. Thurin, J., Thurin, M., Herlyn, M., gen. J. NatLCancerlnst. 74:1047-57 Elder, D. E., Steplewski,Z., Clark, W. 30. Cahan,L. D., Irie, R. F., Singh, R., H., Koprowski, H. 1986. GD2gan-

Annual Reviews

Annu. Rev. Immunol. 1988.6:283-308. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

MELANOMA-ASSOCIATED ANTIGENS glioside biosynthesisis a distinct biochemical event in humanmelanoma tumor progression. FEBSLett. 208: 17-22 40. Natoli, E., Livingston,P. O., Pukel,C. S., Lloyd,K. O., Wiegandt,H., Szalay, J., Oettgen,H. F., Old, L. J. 1986. A murine monoclonalantibody detecting N-acetyl- and N-glycosyl-GM2:characterizationof cell surfacereactivity. CancerRes. 46:4116-20 41. Real, F. X., Houghton,A. N., Albino, A. P., Cordon-Cardo,C., Melamed,M. R., Oettgen, H. F., Old, L. J. 1985. Surface antigens of melanoma and melanocytes defined by mousemonoclonal antibodies: specificity analysis and comparisonof antigenic expression in cultured cells and tissues. CancerRes. 45:4401-11 42. Dippold,W.G., Diener, H. P., Knuth, A., Meyerzum Buschenfelde, K.-H. 1985. Immunohistological localization of ganglioside GD3in humanmalignant melanoma, epithelial tumors, and normaltissues. CancerRes. 45: 36993705 43. Matyas,G. R., Evers, D. C., Radinsky, R., Morre, D. J. 1986. Fibronectin binding to gangliosides and rat liver plasmamembranes. Exp. Ceil Res. 162: 296-318 44. Cheresh,D. A., Klier, F. G. 1986. Disialoganglioside GD2distributes preferentially into substrate-associated microprocesses on human melanoma cells during their attachment to fibronectin. J. Cell Biol. 102:1887-97 45. Cheresh,D. A., Harper,J. R., Schulz, G., Reisfeld, R. A. 1984. Localization of the ganglioside GD2and GD3in adhesionplaquesand on the surface of melanoma cells. Proc. Natl. Acad.Sci. USA 81:5767-71 46. Iliopoulos,D., Ernst, C. S., Steplewski, Z., Jambrosic,J., Rodeck,U., Herlyn, M., Clark, W.H., Koprowski,H. 1987. Inhibition of metastases of a human melanomaxenograft by monoclonal antibody to the GD2/GD3gangliosides. CancerRes. In press 47. Irie, R. F., Sze, L. L., Saxton,R. E. 1982. Humanantibody to OFA-I, a tumor antigen, producedin vitro by Epstein-Barr virus-transformed human B-lymphoid cell lines. Proc.Natl. Acad. Sci. USA79:5666-70 48. Watanabe,T., Pukel, C. S., Takeyama, H., Lloyd,K. O., Shika,H., Li, Id. T. C., Travassos, L. R., Oettgen, H. F., Old, L. J. 1982. Human melanoma antigen AHis an autoantigenic ganglioside related to GD2.J. Exp. Med. 156:

303

1884-89 49. Tai, T., Cahan, L. D., Tsuchida, T., Saxton, R. E., Irie, R., Morton,D. L. 1985. Immunogenicityof melanomaassociated gangliosides in cancer patients. Int. J. Cancer35:607-12 50. Hersey,P., Schibeci, S. D., Townsend, P., Burns, C., Cheresh, D. A. 1986. Potentiation of lymphocyteresponses by monoelonalantibodies to the ganglioside GD3.CancerRes. 46:6083-90 51. Katoh-Semba, R., Facci, L., Skaper,S. D., Varon,S. 1986.Gangliosidesstimulate astroglial cell proliferationin the absenceof serum.J. Cell. Physiol.126: 147-53 52. Saito, M., Terui, Y., Nojiri, H. 1985. Anacidic glycosphingolipid, monosialoganglioside GM3,is a potent physiological inducer for monocyte differentiation of humanpromyelocytic leukemiacell line HL-60cells. Biochem. Biophys. Res. Commun. 132:223-31 53. Koprowski,H., Herlyn, M., Balaban, G., Parmiter, A., Ross,A. H., Nowell, P. C. 1985. Expressionof the receptor for epidermalgrowthfactor correlates with increased dosage of chromosome 7 in malignantmelanoma.Somat.Cell Mol. Genet. 11:297-302 54. Rodeck,U., Herlyn, M., Menssen,H. D., Furlanetto, R. W., Koprowski,H. 1987. Metastaticbut not primarymelanomacells growin vitro independently from exogenousgrowthfactors. Int. J. Cancer.In press 55. Rodeck, U., Herlyn, M., Koprowski, H. 1987. Monoclonalantibodies and growthfactor receptors. J. Cell Biochem.In press 56. Huebner, K., Isobe, M., Chao, M., Bothwell,M., Ross, A. H., Finan, J., Hoxie,J. A., Sehgal,A., Buck,C. R., Lanahan,A., Nowell, P. C., Koprowski, H., Croce, C. M.1986. Thenerve growth factor receptor gene is at human chromosome region 17q1217q22, distal to the chromosome17 breakpoint in acute leukemia. Proc. Natl. Acad. Sci. USA83:1403-7 57. Ross, A. H., Herlyn, M., Maul,G. G., Koprowski,H., Bothwell, M., Chao, M., Pleasure, D., Sonnenfeld,K. 1986. The nerve growth factor receptor in normal and transformed neural crest cells. Ann.N.Y. Acad.Sci. 486:115-23 58. Ross, A. H., Grob, P., Bothwell, M. A., Elder, D. E., Ernst, C. S., Marano, N., Ghrist, B. F. D., Stemp, C. C., Herlyn, M., Atkinson, B., Koprowski, H. 1984. Characterization of nerve growthfactor receptor in neural crest tumors using monoelonalantibodies.

Annual Reviews

Annu. Rev. Immunol. 1988.6:283-308. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

304

HERLYN & KOPROWSKI

Proc. Natl. Acad. Sci.USA81:6681K. O., Old, L. J. 1978. Expressionof 85 Ia-like antigens on cultured malignant 59.Woodbury, R.G.,Brown, J.P.,Loop, melanoma cell lines. Proc. Natl. Acad. S.M.,Hellstrrm, K.E.,Hellstrrm, I. Sei. USA75:6235-39 1981.Analysis ofnormal neoplastic71. Wilson,B. S., Indivieri, F., Pellegrino, human tissues forthetumor-associatedM.A., Ferrone, S. 1979. DR(Ia-like) protein p97. Int. J. Cancer27:145-49 antigens on humanmelanomacells. J. 60. Woodbury,R. G., Brown,J. P., Yeh, Exp. Med. 149:658-68 M.-Y.,Hellstrrm, I., Hellstrfm, K. E. 72. Taramelli,D., Balsari, A., Fossati, G., 1981. Identification of a cell surface Auichini, A., Gambacorti-Passerini, protein, p97, in humanmelanomasand C., Kirkwood,J. M., Parmiani, G. certain other neoplasms.Proc. Natl. 1986. Immuneresponse to autologous Acad. Sci. USA77:2183-87 humanmelanoma:implication of class 61. Brown,J. P., Woodbury,R. R., Hart, I and II MHC products. Biochim. Bitphys. Acta 865:235-51 C. E., Hellstrrm, I., Hellstrrm, K. E. 1981. Quantitativeanalysis of melano- 73. Natali, P., Bigotti, A., Cavalieri, R., ma-associated antigen p97 in normal Nicotra, M. R., Tecec, R., Manfredi, andneoplastictissue. Proc.Natl. Acad. D., Chen, Y.-X., Nadler, L. M., Ferrone, S. 1986. Geneproducts of the Sci. USA78:539-43 62. Brown, J. P., Nishiyama, K., HLA-Dregion in normal and maligHellstrfm, I., Hellstrfm, K. E. 1981. nant tissues of nonlymphoidorigin. Structural characterization of human Hum.Irnmunol. 15:220-33 melanoma-associatedantigen p97 with 74. Reynier, M., Lebec, S., Rohner, C., monoclonalantibodies. J. Immunol. Kalil, J., Aubert,C. 1984.Ia-Likeantigenexpressionon biologicallydifferent 127:539-46 63. Brown,J. P., Hewick, R. M., Hellhumanmelanomacell lines. Eur. J. strrm, I., Hellstrrm,K. E., Doolittle, CancerClin. OncoL20:659-65 R. F., Dreyer, W. J. 1982. Human 75. Taramelli, D., Fossati, G., Mazzocchi, melanoma-associatedantigen p97 is A., Delia, D., Ferrone, S., Parmiani, structurally andfunctionallyrelated to G. 1986. Classes I and II HLAand melanoma-associatedantigen exprestransferrin. Nature296:171-73 64. Rose, T. M., Plowman,G. D,, Teplow, sion and modulation on melanoma D. B., Dreyer,W.J., Heilstrrm,K. E., ceils isolated from primaryand metaBrown,J. P. 1986. Primarystructure of static lesions. CancerRes. 46:433-39 the humanmelanoma-associatedanti76. Ruiter, D. J., Bergman,W., Welvaart, gen p97 (melanotransferrin) deduced K., Scheffer, E., VanVloten, W.V., from the mRNA sequence. Proc. Natl. Russo, C., Ferrone, S. 1984. Immunohistological analysis of malignant Acad. Sci. USA83:1261-65 65. Moore,B. W.1965. A soluble protein melanomas and nevocellular nevi with characteristic of the nervoussystem. monoclonal antibodies to distinct Biochem. Biophys. Res. Commun.19: monomorphic determinants of HLA 739-44 antigens. CancerRes. 44:3930-35 66. Isobe, T., Tsugita, A., Okuyama,T. 77. Broecker, E.-B., Suter, L., Sorg, C. 1978. Theaminoacid sequenceand the 1984. HLA-DR antigen expression in primary melanomasof the skin. J. subunit structure of bovinebrain S 100 protein. J. Neurochem.30:921-23 Invest. DermatoL82:244-47 67. VanEldik,L. J., Zendegin,J. G., Mar- 78. Broecker, E.-B., Suter, L., Brueggen, J., Ruiter, D. J., Macher,E., Sorg, C. shak, D. R., Watterson, D. M. 1982. 1985. Phenotypic dynamics of tumor Calcium-binding proteins and the progression in humanmalignantmelamolecularbasis of calciumaction. Int. Rev. Cytol. 77:I-6 noma.Int. J. Cancer36:29-35 68. Gaynor, R., Irie, R., Morton, D., 79. Guerry,D., Alexander,M.A., Herlyn, Herschrnann, H. R. 1980. S-100 proM., Zelmgebot,L. M., Mitchell, K. F., tein is present in culturedhumanmaligZmijewski,C. M., Lusk, E. J. 1984. HLA-DR histocompatibility leukocyte nant melanomas.Nature 286:400-1 69. Weiss, S. W., Langloss, J. M., antigens permit cultured humanmelaEnzinger, F. M. 1983. Valueof S-100 nomacells fromearly but not advanced protein of soft tissue tumorswith pardisease to stimulate autologous lymticular reference to benignand maligphocytes. J. Clin. Invest. 73: 26771 nant Schwann cell tumors.Lab. Invest. 49:299-308 80. Fossati, G., Taramelli,D., Dalsari, A., Bogdanovich,G., Andreola, S., Par70. Winchester, R. G., Wang, C.-Y., Gibofsky, A., Kunkle, H. G., Lloyd, miani, G. 1984. Primarybut not meta-

Annual Reviews

Annu. Rev. Immunol. 1988.6:283-308. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

MELANOMA-ASSOCIATED ANTIGENS static humanmelanomaexpressing DR antigens stimulate autologous lymphocytes. Int. J. Cancer33:591-97 81. Guerry, D. IV, Alexander, M. A., Elder, D. E., Herlyn, M. 1987. Interferon-gammaregulated the T-cell response to precursor nevi and biologically early melanoma. J. Immunol.139: 305-12 82. Thomson,T. M., Mattes, M., Ranx, L., Old, L. J., Lloyd,K. O. 1985.Pigmentation-associated giycoprotein of human melanomasand melanocytes: definition with a mousemonoclonal antibody.J. Invest. Dermatol.85: 16974 83. Hayashibe,K., Mishima,Y., Ichihashi, M., Kawai, M. 1986. Melanosomal antigenicexpressionon the cell surface andintracellular subunits within melanogenic compartments of pigment cells: analysis by anti-melanosomeassociated monoclonalantibody. J. Invest. Dermatol.87:89-94 84. Maeda, K., Jimbow, K. 1987. Development of MoAbHMSA-2for melanosomesof humanmelanomaand its application to immunohistopathologic diagnosis of neoplastic melanocytes. Cancer59:415-23 85. Akutsu, Y., Jimbow,K. 1986. Development and characterization of a mouse monoclonal antibody, MoAb HMSA-1, against a melanosomalfraction of humanmalignant melanoma. CancerRes. 46:2904-11 86. Atkinson,B. F., Ernst, C. S., Ghrist, B. F. D., Ross, A. H., Clark, W.H., Herlyn, M., Herlyn, D., Maul, G. G., Steplewski, Z., Koprowski,H. 1985. Monoclonalantibody to a highly glycosylatedprotein reacts to fixed tissue with melanomaand other tumors. Hybridoma 4:243-55 87. Donoso,L. A., Folberg, R., Edelberg, K., Arbizzo, V., Atkinson, B. F., Hedyn, M. 1985. Tissue distribution and biochemicalproperties of an ocular melanoma-associatedantigen. Histochem. Cytochem.33:1190-96 88. Vennegoor,C., Calafat, J., Hageman, Ph., van Buitenen, F., Janssen, H., Kolk, A., Rumke, Ph. 1985. Biochemicalcharacterization and cellular localization of a formalin-resistant melanoma-associatedantigen reacting with monoclonal antibody NKI/C-3. Int. J. Cancer35:287-95 89. Sikora, L. K. J., Pinto, A., Demetrick, D. J., Dixon,W.T., Urbanski,S. J., Temple,W., Jerry, L. M. 1987. Characterization of a novelneuroglandular antigen (NGA)expressed on abnormal

305

humanmelanocytes.Int. J. Cancer39: 138-45 90. Ross, A. H., Dietzschold,B., Jackson, D.M., Earley,J. J. Jr., Ghrist,B. F. D., Atkinson, B. F., Koprowski,H. 1985. Isolation and aminoterminal sequencing of a novel melanoma-associated antigen. Arch. Biochem.Biophys. 242: (2) 540-48 91. Ernst, C. S., Shen,J. W.,Litwin, S., Heflyn, M., Koprowski,H., Sears, H. F. 1986. Multiparameterevaluation of the expression of normal and tumorassociated antigens in humancolorectal carcinoma.J. Natl. CancerInst. 77:387-96 92. Natali, P. G., Nicotra,M.R., Bellocci, M.,Cavaliere,R., Bigotti, A. 1985.Distribution of lamininand collagentypeIV in benign and malignantlesions of melanocyteorigin. Int. J. Cancer35: 461-67 93. Nakanishi, T., Hashimoto,K. 1987. Thedifferential reactivity of benignand malignant nevo melanoeytie lesions with mouse monoclonal TNKH1. Cancer 59:1340-44antibody 94. Noronha,A. B., Harper, J. R., Ilyas, A. A., Reisfeld, R. A., Quades,R. H. 1986. Myelin-associatedglyeoprotein shares an antigenic determinantwith a glycoprotein of47:1558-65 humanmelanoma cells. J. Neurochem. 95. Abo,T., Balch, C. M. 1981. A differentiation antigen of humanNKand K cells identified by a monoclonalantibody(HNK-1).J. Immunol.127: 102429 96. Houghton, A. N., Cordon-Cardo,C., Eisinger, M.1986.Differentiationantigens of melanomaand melanocytes. Int. Rev. Exp. Pathol. 28:317-29 97. Herlyn, M., Guerry, D., Koprowski, H. 1985. Recombinant),-interferon induces changes in expression and shedding of antigens associated with normal human melanocytes, nevus cells, andprimaryand metastatic melanomacells. J. Immunol.134:4226-31 98. Tsujisaki, M., Igarashi, M., Sakaguchi, K., Eisinger, M., Herlyn, M., Ferrone, S. 1987. Immunochemicaland functional analysisof HLA class II antigens induced by recombinantimmuneinterferon or normal human epidermal melanocytes. J. Immunol.138: 131016 99. Albino, A. P., Houghton,A. N., Eisinger, M., Lee,J. S., Kantor,R. R. S., Oliff, A. I., Old,L. J. 1986.Class II histocompatibility antigen expression in humanmelanocytestransformed by Harvey murine sarcoma virus (Ha-

Annual Reviews

Annu. Rev. Immunol. 1988.6:283-308. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

306

HERLYN & KOPROWSKI

MSV)and Kirsten MSV retroviruses. J. Exp. Med. 164:1710-22 100. Mancianti, M. L., Herlyn, M.~Well, D., Jambrosic,J., Rodeck,U., Becket, D., Diamond, L., Clark, W. H., Koprowski,H. 1987. Pbenotypiccharacteristics of cultured humannevus cells. J. Invest. Dermatol. In press I01. Tsuchida, T., Saxton, R. E., Morton, D. L., Irie, R. F. 1987.Gangliosidesof humanmelanoma. J. Natl. CancerInst. 78:45-54 102. Holzmann, B., Broecker, E. B., Lehmann, J. M., Ruiter, D. J., Sorg, C., Riethmuller, G., Johnson,J. P. 1987. Tumorprogression in humanmalignant melanoma: five stages defined by their antigenicphenotypes.Int. d. Cancer 39:466-71 103. Brneggen,J., Sorg, C. 1983. Detection of phenotypic differences on human malignant melanomalines and their variant sublines with monoclonalantibodies. CancerImmunol.Immunother. 15:200-5 104. Lehmann,J. M., Holzmann,B., Breitbart, E. W., Schmiegelow,P., Riethmuller, G., Johnson,J. P. 1987. Discrimination between benign and malignant cells of the melanocytic lineage by two novel antigens, gpll3 and p76. CancerRes. 47:841-45 105. Johnson, J. P., Demmer-Dieckmann, M., Meo, T., Hadam,M. R., Riethmuller, G. 1981. Surface antigens of human melanoma cells defined by monoclonalantibodies. 1. Biochemical characterization of two antigens found on cell lines andfresh tumorsof diverse origin. Eur. J. ImmunoL 11:825-31 106. Ruiter, D. J., Dingjan,G. M., Steulen, P. M., van Beveren-Hoover, M., de Graaf-Reitsma, C. B., Bergman,W., van Muijen, G. N. P., Warnaar,S. O. 1985. Monoelonalantibodies selected to discriminate between malignant melanomasand nevocellular nevi. J. Invest. DermatoL85:4-8 107. Holzmann,B., Johnson, J. P., Kaudewitz, P., Riethmuller,G. 1985.In situ analysis of antigens on malignantand benigncells of the melanocytelineage. J. Exp. Med. 161:366-77 108. Atkinson,B., Ernst, C. S., Ghrist, B. F. D., Herlyn, M., Herlyn, D., Ross, A. H~, Blaszczyk,M., Steplewski, Z., Koprowski, H. 1984. Fixed tissue screening of monoclonalantibodies against humanmalignant melanoma. CancerRes. 44:2577-81 109. Herlyn,M., Lange,B., Bennicelli, J., Blaszczyk,M., Guerry,D., Koprowski, H.1984.Increasedlevels of circulating

HLA-DR antigen in sera of patients with lymphoblastoidleukemias. Leukemia Res. 8:323-34 110. Ross,A. H., Herlyn, M., Ernst, C. S., Guerry,D., Bennicelli,J., Gbrist,B. F. D., Atkinson, B., Koprowski,H. 1984. Immunoassayfor melanoma-associated proteoglycanin sera of patients using monoclonaland polyclonal antibodies. CancerRes. 44:4642-47 111. Giacomini,P., Veglia,F., Cordiali Fei, P., Rehle,T., Natali, P. G., Ferrone,S. 1984. Level of membrane-bound highmolecular-weight melanoma-associated antigen and a cytoplasmic melanoma-associatedantigen in surgically removedtissues and sera frompatients with melanoma.CancerRes. 44: 128187 112. Giacomini,P., Aguzzi,A., Pestka, S., Fisher, P. B., Ferrone, S. 1984. Modulation by recombinantDNAleukocyte (~) andfibroblast(/~) interferonsof expression and shedding of HLA-and tumor-associated antigens by human melanoma cells. J. Immunol.133: 164955 113. Donoso, L. A., Felberg, N. T., Edelberg,K., Bodinghaus,P., Herlyn, M. 1986. Metastatic uveal melanoma: an ocular melanoma-associatedantigenin the serumof patients with metastatic disease. J. Imrnunoassay 7: 27383 114. Morgan,A. C., Crane, M. M., Rossen, R.-D. 1984. Measurementof a monoclonal antibody-defined, melanomaassociated antigen in humansera: correlation of circulating antigen levels with tumor burden. J. Natl. Cancer Inst. 72:243-49 115. Schulz, G., Cheresh,D. A., Varki, N. M., Yu,A., Staffelino, C. K., Reisfeld, R. A. 1984. Detection of ganglioside GD2in sera and tumor tissues of neuroblastomapatients. CancerRes. 44:5914-20 116. Ladisch,S., Wu,Z.-L., Feig, S., Ulsh, L,, Schwartz,E., Flautsis, G., Wiley, F,, Lenarsky,C., Seeger,R. 1987.Shedding of GD2ganglioside by human neuroblastoma.Int. ,1. Cancer39: 7376 117. Dillmann, R. O., Beauregard, J. C., Sobol, R. E., Royston, I. R., Bartholomew, R. M., Hagan, P. S., Halpern, S. E. 1984. Lack of radioimmunodetection and complications associatedwith cross-reactivity withan antigenoncirculating cells. CancerRes. 44:2213-18 118. Herlyu,D., Powe,J., Alavi,A., Mattis, J., Herlyn, M., Ernst, C. S., Vaum,

Annual Reviews

Annu. Rev. Immunol. 1988.6:283-308. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

MELANOMA-ASSOCIATED ANTIGENS R., Koprowski,H. 1983. Radioimmunodetection of humantumor xenografts by monoclonalantibodies. Cancer Res. 43:2731-35 119. Herlyn, D., Powe, J., Munz,D. L., Alavi, A., Herlyn, M., Srivastava, S. C., Koprowski, H. 1986. Radioimmunodetection of human tumor xenografts by monoclonal antibody F(ab’)2 fragments. Int. J. Nucl. Med. Biol, 13:401-5 120. Powe,J., Herlyn, D., Alavi, A., Munz, D., Steplewski, Z., Koprowski, H. 1987. Radioimmunodetectionof human tumor xenografts by monoclonal antibodiescorrelates with antibody density and affinity. In lmmunoscinti#raphy,ed. K. Button, L. Donato, pp. 139-56. NewYork: Gordon& BreachSei. 121. Douillard,J. Y., Lehur,P. A., Aillet, G., Kreiner, M., Bianco-Arco, A., Peltier, P., Chatal, J. F. 1986. Immunohistochemicalantigenic expression and in vivo tumor uptake of monoclonal antibodies with specificity for tumorsof the gastrointestinal tract. CancerRes. 46:4221-24 122. Berche,C., Mach,J. P., Lumbroso, J.D., Langlais, C., Aubry,F., Buchegger, I., Carrel, S., Rougier,P., Parmentier, C., Tubiana, M. 1982. Tomoscintigraphyfor detecting gastrointestinal and medullarythyr.oid cancers: first clinical results using radiolabeled monoclonalantibodies against carcinoembryonicantigen. Br. Med. J. 285: 1447-51 123. Larson,S. M., Brown,J. P., Wright,P. W.,Carrasquillo,J. A., Hellstrtm,1., Hellstrtm, K. E. 1983. Imaging of melanomawith 13q-labeled monoclonal antibodies. J. Nucl, Med.24: 123-29 124. Halpern, S. H., Dillman, R. O., Witztum,K. P., Shega,J. P., Hagan,P. L., Burrows, W.M., Dillman, J. B., Clutter, M.L., Sobol, R. E., Frincke, J. M., Bartholomew, R. M., David, G. S., Carlo, D. J. 1985. Radioimmunodetection of melanomautilizing In-’’1 96£ monoclonalantibody:a preliminary report. Radiology155:493-99 125. Murray, J. L., Rosenblum, M. G., Sobol, R. E., Bartholomew,R. M., Plager, C. E., Haynie,T. P., Jahns, M. F., Glenn,H. J., Lamki,L., Benjamin, R. S., Papadopoulos,N., Boddie, A. W.,Frincke,J. M., David,G. S., Carlo, D. J., Hersh, E. M. 1985. Radioimmunopagingin malignant melanoma with ~’ln-labeled monoclonal antibody96.5. CancerRes. 45:2376-81

307

126. Buraggi,G. L., Callegaro,L., Mariani, G., Turrin,A., Cascinelli,N., Attili, A., Bombardieri,E., Terno, G., P1assio, G., Dovis, M., Mazzuca,N., Natali, P. G., Scassellati, G. A., Rosa, U. Ferrone S. 1985. Imaging with 3q-labeled monoclonal antibodies to a high-molecular-weightmelanomaassociated antigen in patients with melanoma:efficacy of whole immunoglobulinand its F(ab’)2 fragments. CancerRes. 45:3378-87 127. Buraggi,G. L., Callegaro,L., Turrin, A., Cascinelli,N., Attili, A., Emanuelli, H., Gasparini,M., Deleide,G., Plassio, G., Dovis,M., Mariani,G., Natali, P. G., Scassellati, G. A., Rosa, U,, Ferrone. S. 1984. Immunoscintigraphy with 1251, 99mTcand Uqn-labeled F(ab’)2 fragmentsof monoclonalantibodies to human high molecular weight-melanomaassociated antigen (HMW-MAA). d. NucL Med. Allied Sci. 28:283-95 128. Siccardi, A. G., Buraggi, G. L., Callegaro, L., Mariani,G., Natali, P. G,, Abbati, A., Bestagno,M., Caputo,V,, Mansi, L., Masi, R., Paganelli, G,, Riva, P., Salvatore,M., Sanguineti,M., Troncone, L., Turco,G. L,, Scassellati, G. A., Ferrone, S. 1986. Multicenter study of immunoscintigraphy with radiolabeled monoclonalantibodies in patients with melanoma.CancerRes. 46:4817-22 129. Eger, R. R., Covell, D. G., Carrasquillo, J. A., Abrams,P. G., Foon,K. A., Reynolds,J. C., Schroff, R. W., Morgan, A. C., Larson, S. M., Weinstein, J. N. 1987. Kinetic model for the biodistribution of an rainlabeled monoclonal antibody in humans. CancerRes. 47:3328-36 130. Herlyn, D., Koprowski, H. 1982. IgG2amonoclonalantibodies inhibit humantumor growth through interactions witheffector cells. Proc.Natl. Acad. Sci. USA79:476245 131. Herlyn, D., Lubeek,M.D., Steplewski, Z., Koprowski,H. 1985. Destructionof human tumors by IgG2a monoclonal antibodies and macrophages.In Monoclonal Antibodies and CancerTherapy, ed. R. A. Reisfeld,S. Sell, 27: 165-72. NewYork: Liss 132. Steplewski, Z., Lubeck, M. D., Koprowski, H. 1983. Humanmacrophages armed with murine immunoglobulin ~2a antibodies to tumors destroy humancancer cells. Science 221:865-67 133. Herlyn,D., Herlyn,M., Steplewski,Z., Koprowski,H. 1985. Monoclonalanti-

Annual Reviews

Annu. Rev. Immunol. 1988.6:283-308. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

308

HERLYN & KOPROWSKI

humantumorantibodies of six isotypes Cordon-Cardo, C., Welt,S., Fliegel, B., in cytotoxic reactions with humanand Vadham,S., Carswell, E., Melamed, murineeffector cells. Cell Immunol.92: M.R., Oettgen,H. F., Old, L. J. 1985. 105-14 Mouse monoclonal IgG3 antibody 134. Hellstr6m,I., Brown,J. P., Hellstr6m, detecting GD3ganglioside: a phase I K. E. 1981. Monoclonalantibodies to trial in patients with malignantmelatwo determinants of melanomaantigen noma.Proc. Natl. Acad. Sci. USA82: p97 act synergistically in complement1242-46 dependentcytotoxicity. J. Immunol. 140. Goodman,G. E., Beaumier,P., Hell127:157-60 strfm, I., Feryhough,B., Hellstr6m, 135. Schulz, G., Bumol,T. F., Reisfeld, K. E. 1985. Pilot trial of murine R. A. 1983. Monoclonal antibodymonoclonalantibodies in patients with directedeffector cells selectively lyse advanced melanoma.J. Clin. Oncol. humanmelanoma cells in vitro and in 3:340-52 vivo. Proc. Natl. Acad. Sci. USA80: 141. Oldham,R. K., Foon, K. A., Morgan, 5407-11 A. C., Woodhouse, C. S., Schroff, R. 136. Matzku,S., Broecker,E. B., Brueggen, W., Abrams,P. G., Fer, M., SchoenJ., Dippold, W.G., Tilgen, W.1986. berger, C. S., Farell, M., Kimball,E. Modesof binding and internalization 1984. Monoclonalantibody therapy of of monoclonal antimelanoma antimalignant melanoma:in vivo localbodies. CancerRes. 46:384-85 ization in cutaneousmetastatis after 137. Cheresh,D. A., Housik,C. J., Staffiintravenous administration. J. Clin. leno, C. K., Jung, G., Reisfeld, R. A. Oncol. 2:1235-44 1985. Disialoganglioside (3D3on hu- 142. Schroff, R. W., Woodhouse,C. S., man melanomaserves as a relevant Foon, K. A., Oldham,R. K., Farell, target antigen for monoclonalantiM. M., Klein, R. A., Morgan,A. C. body-mediatedtumor cytolysis. Proc. 1985. Intratumor localization ofmonoNatl. Acad. Sci. USA82:5155-59 clonal antibodyin patients with mela138. Houghton,A. N., Schienberg, D. A. noma treated with antibody to a 1986. Monoclonalantibodies: poten250,000 dalton melanoma-associated tial applications to the treatment of antigen. J. Natl. CancerInst. 74: 299cancer. SeminarsOncol. 13:165-79 306 139. Houghton, A. N., Mintzer, D.,

Annu. Rev. Immunol. 1988.6:283-308. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Annu. Rev. Immunol. 1988.6:283-308. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Annual Reviews www.annualreviews.org/aronline Ann. Rev. Immunol.1988. 6: 309-26 Copyright©1988by AnnualReviewsInc, All rights reserved

Annu. Rev. Immunol. 1988.6:309-326. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

THE DEVELOPMENTAL BIOLOGY OF T LYMPHOCYTES 1HaraM yon Boehmer Basel Institute for Immunology, Grenzacherstrasse487, Postfach, CH-4005Basel, Switzerland T LYMPHOCYTES T lymphocytesmorphologically similar to B lymphocytesalso express surface receptorsfor antigenthat are subject to allelic exclusion.Receptor gene DNA segments of both lymphocyteclasses undergo rearrangement in somatic cells. OtherwiseT lymphocyteshave little in common with B lymphocytes.The preoccupation of immunologistsover the decades with the easily accessible immunoglobulins has resulted in obscuring rather than revealingthe nature of antigenrecognitionby T cells (1). Thecloning of T cells (2), the identification of their receptors(3-9), andthe analysis of their function(2, 10) haveput an endto the myriadof false andsenseless analogies attemptingto equate the two majorclasses oflymphocytes.Some uniquefeatures of T lymphocytesrelevant to their developmentalbiology serve to introduce this class of lymphocytesto the less experienced reader. Surface

Markers Definin9

Lymphocyte Subsets

MurineT lymphocytesexpress the Thyl antigen, a 24-kd glycoprotein identified by a numberof monoclonalantibodies (11). An even better markerto distinguish T lymphocytesfromthe remaininghemopoieticcells is the CD3complexconsisting of at least six different proteins associated with T cell receptors for antigen (12, 13). Like Thyl, the murineCD3 complexcan be identified by a recently developedmonoclonalantibody (14). Peripheral T lymphocytes maybe divided into subpopulations ~Onsabbatical leave at the Massachusetts Institute of Technology, Cancer Research Center, 40 AmesStreet, Cambridge, Massachusetts 02139.

309 0732-0582/88/0410-0309502.00

Annual Reviews 310

VONBOEHMER

according to their expression of CD4(15, 16) and CD8(17, 18) proteins; approximately two thirds express CD4 but not CD8, and one third CD8but not CD4proteins. In this paper, these cell types are referred to as single positive CD4÷, CD8-as well as CD4-, CD8÷ cells. A very small proportion of peripheral CD3÷ cells expresses neither CD4nor CD8.

Annu. Rev. Immunol. 1988.6:309-326. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Function

and Specificity

of Lymphocyte Subsets

Unlike antigen receptors of B cells, antigen receptors of T cells as a rule are not secreted and do not bind soluble antigens like bacterial toxin (19). cell receptors recognize peptides associated with major histocompatibility complex (MHC)molecules on the cell surface (20-22). MHCmolecules proteins encoded by genes of the MHC,which~represents a chromosomal region on mouse chromosome17 (23). Twoclasses of MHCmolecules are important here, namely, the class I MHCproteins expressed on most nucleated cells and the class II MHC proteins with a more restricted tissue distribution expressed on B lymphocytes, macrophages, and dendritic cells and also on epithelial cells of the thymus. Usually, class I proteins associate with peptides present in the cytosol (24, 25). These peptides may be degraded products of viral proteins virally infected cells (24) or of other proteins present in the cytosol. By unknownmechanismthey are transported to the cell surface. Class II proteins bind peptides derived from external proteins which are endocytosed by macrophages,dendritic cells, or B lymphocytes and are cleaved in lysosomes (21, 22). Antigen receptors on the CD4÷, ÷) CD8- (CD4 subset of T lymphocytes bind class II MHCantigen associated peptides; receptors on the CD4-, CD8÷ (CD8÷) subset bind class I MHCantigen ÷ cells develop associated peptides. After antigenic stimulation the CD4 into helper cells, helping B cells to makeantibodies or helping the clonal ÷ cells which after antigenic stimulation develop into expansion of CD8 + and of CD8 + cells are not cytolytic T cells. The functional potential of CD4 entirely distinct; both classes are able to produce several lymphokinesand ÷ cells have not been obtained directly to becomecytolytic. Cytolytic CD4 from animals but can develop in vitro. The most important lymphokines produced by T lymphocytes are interleukin 2 (26, 27) and interleukin (28, 29). Also y-interferon is produced by these cells (30). T lymphocytes produce not only lymphokines; after antigenic stimulation, they also express receptors which bind these lymphokines, thereby regulating their growth and differentiation. The best characterized receptor is the IL-2 receptor (31) expressed by both activated CD4÷ and CD8÷ lymphocytes. The antigen specificity of the peripheral CD3+, CD4-, CD8- cells is unknown.It has been argued that they do not recognize MHCassociated

Annual Reviews T LYMPHOCYTE DEVELOPMENT

311

Annu. Rev. Immunol. 1988.6:309-326. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

antigens (32, 33). Thoseclaims, however,are largely unsubstantiated becausethey are based on experimental conditions under whichthe MHC ÷ cells can also be obscured,i.e. whereCD8 ÷ cells kill specificity of CD8 nonspecificallya variety of different targets (34). Likethe single positive cells, the doublenegative CD3÷ cells can be inducedto produceIL-2 and becomecytolytic (35). Antigen Receptors and Accessory Molecules ++ Onemight have expected that the antigen receptors on CD4 and CD8 cells are encoded bydifferent loci becauseof the differentantigenspecificity + and CD8 + cells both of these cells. However,this is not the case: CD4 express ~, fl heterodimersencodedby genes of the sameloci, and in fact + as well as CD8 + cells (9). the same gene segmentscan be used by CD4 Earlier it wasshownthat these ~, fl receptors are necessaryfor antigen recognition(36), andmorerecent experimentsindicate that these receptors are the only variable clonotypic moleculesmediatingthe specificity for MHC antigen and peptides, i.e. the MHC restricted antigen specificity. Suchspecificity can be transferred fromone T cell clone to another by transfecting just the productivelyrearranged~ andfl genes(9, 37-39). The ~ and fl genes each encodea protein of about 40 kd. Thetwo chains are expressed on the cell-surface as a disulfide linked heterodimerand are associated with the CD3complex.Since the ~, fl heterodimeris the only + cells are class II clonotypic molecule,the question arises as to whyCD4 ÷ and CD8 class I MHC antigen specific. Theexplanation maylie with the invariant CD4and CD8molecules themselves. Initial experimentssuggestedthat these moleculesassisted the interaction of T cells with other cells becauseantibodiesdirected against these structures interfered with both the activation and effector function of ÷ or CD8 ÷ cells (40, 41). These experiments were, however, inconCD4 clusive becauseboth antibodies could deliver negative signals to T lymphocytesin the absenceof antigen recognition (42, 43). Direct evidence for the participation of CD8moleculesin the interaction of T cells with other cells was then obtained by transfection experiments: the MHC restricted antigen recognition by an a, fl heterodimerof a CD8-T cell clone could be markedlyenhancedor evenrevealed by transfection of the CD8geneinto that clone (39, 44). It has been argued that the invariant CD4and CD8moleculesbind to invariant proteins of class I and class II MHC antigen, respectively (17, 45). Byitself, this postulationwouldnot be a sufficient explanationfor the correlation of T cell specificity and CD4/CD8 phenotype.If one, howeve~r, assumesthat the crucial signal in the activation of T cells is the crosslinking of~, fl heterodimerson the one handand CD4or CD8proteins on

Annual Reviews

Annu. Rev. Immunol. 1988.6:309-326. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

312

VON BOEt-IMER

the other hand by the sameMHC molecule(46-48), one has an appropriate model.In that case T cells expressingan ~, fl heterodimerfor a class I boundpeptide and ~2D4proteins, as well as those expressing an ~, fl heterodimerfor a class II boundpeptide and CD8proteins, could not be activated becausethat constellation doesnot allow cross-linking of ~, fl and CD4/CD8 proteins by the same MHC molecule. One might argue that only in those situations wherethe ~, fl protein has a very high affinity for antigen is cross-linking of ~, fl and CDproteins not required for T cell activation. In that case the rule of correlation of CD4and CD8phenotype andT cell specificity can be broken. ÷ lymphocytes express not ~, fl heterodimers SomeCD4-, CD8-, CD3 but a different ~5 heterodimerassociated with CD3(49, 50). The7 protein is encoded by the ~ locus consisting of rearranging V, J, and C gene segments(51, 52). The~ locus is located betweenJ~ and V~segmentsand consists of C and J segmentsto whichV~segmentsare joined (53). The biological significance of this interesting geneconstellation needsto be explored. In some mice, especially autoimmunemousestrains, CD4-, CD8-, ÷ cells expressing ~, fl heterodimersare found in the periphery (54, CD3 55). Thebiological significanceof these cells remainsto be elucidated. Allelic

Exclusion of T Cell Receptors for Antiyen

Variousexperimentshaveaddressedthe issue of allelic exclusionof T cell receptors for antigen. A monoclonal antibodydetecting fl proteins encoded by the Vfl8 genefamily distinguishedmousestrains possessingand lacking these genes(56, 57). In positive strains about 20%of the T cells stained with this antibody, while noneof the T cells wasstained in the negative strains. Typicallya heterozygousF1 hybridbetweena positive and negative strain possessed10%of T cells reacting with the antibody. This result was interpreted to reflect allelic exclusion (56). When¯ and fl alleles were analyzedin T cell clones, it wasfoundeither that one allele wasincompletely rearranged,i.e. Dto J, or that a VDJjoining of oneallele resulted in a prematurestop codon(37; and H. von Boehmer,Uematsu,Steinmetz, unpublisheddata). Allelic exclusion of T cell receptors maynot be stochastic process, accordingto recent experimentswith fl genetransgenic mice. In these micea productivelyrearrangedfl transgeneexpressedin all T cells prevented further rearrangementof endogenous fl genes; that is, endogenousgenes either did not rearrange at all or rearranged incompletely, i.e. D to J (Uematsu, Ryser, Dembic, Berns, Krimpenfort, Borgulya,von Boehmer,Steinmetz, manuscriptin preparation). The mechanism of this phenomenon is at present not clear. It mayinvolve inaccessibility of Va but not D~gene segmentsto a recombinaseand/or an

Annual Reviews T LYMPHOCYTE DEVELOPMENT 313

earlier rearrangementof the 0t locus such that there is less time for fl rearrangement in the transgenic mice before the recombinase becomes inactive whenboth fl and ~ are productivelyrearranged.

Annu. Rev. Immunol. 1988.6:309-326. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

THYMOCYTES Thevast majority of peripheral T cells originates in the thymuswhereT lymphocytesare formedfrom moreprimitive hemopoieticprecursor ceils whichenter the thymusthroughout life. Small numbersof T lymphocytes maydifferentiate ectopically and account for the T cells found in thymuslessmice. Surface Markers Defining

Thymocyte Subsets

At present weonly need to knowtwo surface markersin addition to those already described for a biologically meaningfuldescription of thymocyte subpopulations.Onemarkeris identified by the J 11 d antibody(58) binding predominantlyan antigen associated with a 50-kd protein. The other antigen is Pgpl (59) probablyexpressedon cells homingto the thymus. Thethymuscontains cells quite similar to peripheral T cells, namely, ÷ and CD8 ÷ cells expressing CD3associated ~, fl single positive CD4 heterodimers. These cells, however, do not comprise morethan 15%of all thymocytes.Byfar the mostabundantcell type is representedby double +, CD8 ÷ thymocytes accounting for approximately 80%of positive CD4 all thymocytes.At least 50%of the double positive cells, whichunlike mostsingle positive cells are J1 ld +, express CD3moleculesat relatively low density on the cell surface (60). The 5%doublenegative CD4-,CD8÷ cells can be dividedinto at least three different subsets: the CD3-,J1 ld +, ~6+, Jlld + cells (60), and the CD3 +, ~fl+, Jlld- cells. cells, the CD3 Thelatter is present in the morematurebut not embryonicthymus(Tables 1, 2) (61). The Functional Potential of Thymocyte Subsets ++ Like their peripheral counterparts the single positive CD4 and CD8 thymocytescan be inducedby antigen or lectin to becomehelper or killer Table 1 Thymocytesubpopulations Singlepositive

Doublepositive

CD4+, CD8- (8%) CD4÷, CD8÷, CD3+ (40%) CD4-, CD8÷ (6%) CD4+, CD8+, CD3- (40%) Total 14%

Total 80%

Doublenegative CD4-, CD8Total 5%

Annual Reviews 314

VON BOEHMER Table2 Doublenegative CD4-,CD8-subpopulations Receptornegative

Annu. Rev. Immunol. 1988.6:309-326. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

+ CD3-,J1 ld 70%

),~5receptor positive +, + CD3 J1 ld I0°/~

~fl receptorpositive ÷, J1 ldCD3 20%

cells. The frequency of inducible cells in this population is somewhatless than that of peripheral T cells (62) and may indicate that functional competenceis acquired after the cells have switched to this phenotype. Neither antigen nor lectin induces significant proliferation or differentiation in double positive CD4÷, CD8+ thymocytes even though 50% of them express CD3molecules. It was noted relatively early that some double negative cells could be induced by lectins and lymphokines to develop into double negative cytolytic T cells in vitro (63). Morerecently, this property has been assigned to the CD3÷, 7~÷ double negative cells (35). The CD3-, Jlld ÷ double negative cells are cycling in vivo (64). proportion of them express proteins detected by monoclonal antibodies directed against one of the chains of the high affinity IL-2 receptor (65, 66). These cells do not proliferate significantly in vitro whencultured in IL-2 or even with the lectin concanavalin A plus IL-2 (67). Proliferation can, however, be induced by the combination of the phorbol ester PMA, the calcium ionophore ionomycin and IL-2 (68, 69) as well as the combination of PMAand IL-4 (70). Whether the lymphokines IL-2 and IL-4 are involved in the in vivo proliferation of these cells is not clear: it was found that embryonic thymocytes can be induced to produce IL-2 (68, 69), and some authors found that anti-IL-2 receptor antibodies inhibit proliferation and differentiation of these cells in organculture (71). Others, however,found that IL-2 can inhibit cell proliferation in organ culture (72). These cells also reportedly cannot endocytose IL-2 (73), a step thought be essential in the initiation of IL-2 induced cell division. Thyrnic

Miyrants

Our information on thymic immigrants is mostly based on the analysis of hemopoietic cells immediately after their entry into the thymus. The phenotype is double negative CD4-, CD8- as well as Thyl, CD3, and IL2 receptor negative. These cells express Pgpl (74) and represent a very minor subset in the adult thymus (75). The thymic emigrants have been characterized by labeling them intracellularly in the thymus with fluorescein (76) and analyzing them immediately after they leave the thymus. These studies suggest that most of the

Annual Reviews

Annu. Rev. Immunol. 1988.6:309-326. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

T LYMPHOCYTE

DEVELOPMENT

315

+ or CD8 + cells with a cells leaving the thymusare single positive CD4 phenotypesimilar to that of medullary thymocytes. It has been argued (77) that medullarycells do not leave the thymus.Thereasons given are that thymic emigrants stained in suspension express high levels of an antigen recognized by the MEL14 antibody, while with section staining, medullarycells do not express MEL14. In a morecareful study, however, about half of the single positive thymocytes,the vast majority of which are located in the medulla, stain with the MEL14 antibody in suspension (78). Until confronted with morecompelling data, we mayassumethat medullarycells, with a phenotypevery similar but not identical to that of peripheral T cells, are leaving the thymus.If double positive or double negative cells are leaving the thymus,they represent a minorfraction of the 2 x 10 6 emigrants leaving the youngadult thymusevery day. PRECURSOR PRODUCT RELATIONSHIP THYMOCYTE SUBPOPULATIONS

OF

Fromthe abovewecan concludethat the thymuscontributes to the pool of peripheral T cells by continuouslyreleasing migrants with relatively mature phenotype. Thus, not muchmaturation occurs of the emigrants in the periphery, a viewopposedto that held by several investigators previously (79, 80). Thereforethe importantevents of T cell differentiation mustoccur pre- or intra-thymically. Avariety of experimentalapproaches havebeen devised to delineate the path of T cell differentiation in the thymus,and the followingrepresents by no meansan exhaustive description of the crucial experiments. Turnover of Cortical and Medullary Cells Morethan 20 years ago it wasnoted that cells in the thymusturned over very rapidly but did not live very long. Up to 20 thymusescontaining rapidly dividingcells weretransplantedinto mice, but the total numberof lymphocytesin the transplanted animals did not increase significantly (81). Continuous3H-thymidine labeling experimentsrevealed that not all thymoeytesubpopulationswereturning over at the samerate. Mostcells of cortical phenotype,i.e. doublepositive cells, wereentirely renewed within three days, i.e. 100%becamelabeled in a three-day period. Onthe other hand,cells of medullaryphenotype,i.e. single positive cells, labeled muchmore slowly (82). Taking into account the cell numbersin both compartments, the daily productionrate is 5 x 10 7 for the doublepositive 6 and2 x 10 for the single positive cells. Mostcortical cells therefore do not becomemedullarycells, and since they are not leaving the thymusin

Annual Reviews

316

VONBOEHMER

significant numbers,they must die intrathymically. This view has been supportedby independentevidence(83).

Annu. Rev. Immunol. 1988.6:309-326. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

The Precursors of Cortical and Medullary Cells Theprecursorsof the cortical doublepositive andmedullarysingle positive cells are amongthe CD4-,CDS-,J1 ld + cells whichare the predominant cell population in the early embryonicthymus.This wasshownby removing the thymusat 14 days of gestation andculturing it for several days in vitro whichprecluded the immigrationof a second waveof moredifferentiated cells (63). In suchcultures the appearanceof single positive CD4-, + and double positive CD4 +, CD8 ÷ (both nonfunctional) cells could CD8 ÷ cells were be monitored in vitro. The early nonfunctional CD4-,CD8 +, + shownto acquire the CD4 CD8 phenotype within 12 hrs of in vitro ++ culture (84). Theearly single positive CD4-,CD8 cells whichare J1 ld + (all embryoniccells are J1 ld at that stage) are also found in the adult thymus(85). Functional single positive cells appearedalways after the nonfunctionaldoublepositive cells in in vitro organculture. This is important becauseif they hadbeendetected beforethe doublepositive cells, that wouldhaveprecludedthe possibility that exclusivelydoublepositive cells are the immediate precursorsof functionalsingle positive cells (see below). Double negative CD4-, CD8-, CD3-, J1 ld + lymphoblasts have also beenisolated fromthe adult thymus(86); in adoptivetransfer experiments it wasshownthat these cells like their embryoniccounterparts possessed precursor potential for the various thymocytes.Thus, the generation of cortical and medullarycells is probablya continuingintrathymicprocess, even in adult mice wherecell production declines to someextent after sexual maturity. Dothe double negative thymic lymphoblastscontain single precursor cells able to give rise to both cortical andmedullarycells? This question has been approachedby repopulating the thymuswith hemopoieticcells in vivo and in vitro with someambiguityin the results. Repopulationwith low numbersof bonemarrowcells in vivo has resulted in thymuseswhich at a certain time werecolonizedin either the medullaalone or both the medullaand the cortex (87). Onthe other hand, colonization with single cells in vitro has providedevidencethat a single intrathymieprecursorcell can give rise to both doublepositive and single positive thymocytes(88). Thelatter evidenceappears conclusivewhile the formermaybe criticized on the groundsthat the animals have only been analyzedat one point in time. Onemight argue that at an earlier time progeny mayhave been detected in all experimentsin both the cortex and the medulla,whichshow vastly different turnover rates of lymphocytes.In summary,the double negative J 11 d+ lymphoblastsof the thymuscontain all the precursors for

Annual Reviews

Annu. Rev. Immunol. 1988.6:309-326. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

T LYMPHOCYTE DEVELOPMENT 317

the other thymocytesubpopulations, and a single intrathymic precursor cell can produceprogenyof both cortical double positive and medullary single positive phenotype.It remainsto be seen whetherthere exist some precursorsfor medullarycells only in extrathymictissue. The Immediate Precursor of Single Positive, Functional Thymocytes Theimmediateprecursors of the slowly turning over single positive thymocytesare difficult to determine becausethey could be derived from either the rapidly dividing doublenegative, doublepositive, or even the late appearingJ 11 d-, doublenegativecells. Thelatter possibility doesnot seemvery likely becausesingle positive J1 ld- cells appear in ontogeny prior to or at the sametime as doublenegative J1 ld- cells and because doublenegative J1 ld- cells showlittle capacity to repopulate any compartment of the thymus(61). Attemptshave been madeto purify double positive cells andto inject themintrathymically,to see whetherthese cells can producesingle positive progeny.Thecrux with that experimentis that most of the double positive cells die anyway,and single positive cells mayalways be generated from tiny contaminantsof the double positive population.Purification of the doublenegativecells is also of little use becausethey alwaysgenerate nonfunctionaldoublepositive cells before functionalJ1 ld- single positivecells. Acleverly designedexperimenthas addressedthe question of precursors for single positive cells in a different way.Stemcell-reconstituted animals ÷, were continuously injected with CD8antibodies, and the numberofCD4 CDS-single positive cells wasdeterminedlater; this addressedthe question ÷, CD8-cells were derived from CD8 ÷ precursors. The of whether CD4 experimentemployedtwo different sets of stemcells whichgaverise to T cells expressingdifferent CD8and Thylalleles. Theinjected CD8antibody was allele specific; and it waspossible, by doublestaining with CD4as well as allele specific Thylantibodies, to determinefromwhichstemcells ÷ cells were derived. In this way, it could be the single positive CD4 + cells occurred only determinedwhether the observedreduction of CD4 in the lineage of cells expressingthe CD8allele against whichthe injected antibodies were directed. This design excludes any nonspecific feeder or ÷ cells on single positive CD4 ÷ cells as a cell interaction effect of CD8 + cell numpossible interpretation for the observedresult of reducedCD4 ÷ cells are derived bers. Thus,this experimentarguesthat at least someCD4 ÷ precursors (89). from CD8 The Precursors of Double Negative CD3÷ Cells ÷, Jlld ÷ cells probably are derived directly from Doublenegative CD3 double negative cells, since they appear early in ontogeny and sim-

Annual Reviews

318

VONBOEHMER

ultaneously with doublepositive cells (60). Theprecursors of the late ÷, J1 ld- cells are unknown,but I argue appearing double negative CD3 bdowthat they are derived from double positive thymocytes.

Annu. Rev. Immunol. 1988.6:309-326. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

THE INTRATHYMIC ACQUISITION RECEPTORS FOR ANTIGEN

OF T CELL

Theearly J1 ld + doublenegative cells present in embryonicthymusdo not express antigen receptors. T cell receptor proteins could not be detected by immunoprecipitation or in situ staining using appropriate antisera and monoclonalantibodies (90, 91). In addition, full length/~ or ct RNA could not be found in these cells (92, 93). The intrathymic acquisition of/~ proteins could be visualized in organculture:/~ protein expressingcells first appeareddispersedandin isolation, while clusters of//positive cells weredetected at a later stage (90). Theseexperimentsdearly refute the viewthat a very smallproportionof/~ expressingcells presentat the onset of cultures were expanding. Rather, the experiment indicates de novo expression of protein after entry of hemopoieticcells into the thymus. Consistent with that view is the increase in/~ DNA rearrangementevident by new bands and the diminishing of germline bands in Southern blot analysis as well as the stepwiseexpressionof first full-length/~ andthen ~ transcripts (92-94). The appearanceof new/~DNA bands has in fact also beenobservedin thymusesreconstituted with a single cell (95). Whilect, /~ T cell receptors for antigen are absent from Jlld ÷, double negative lymphoblasts,they are clearly expressedon doublepositive cortical cells (90, 91), late appearingJ1 ld- doublenegativecells (61, 96), andof course also on single positive medullarycells. In contrast to 0~,/~ heterodimers,y, 6 heterodimersare expressedrelatively early on J1 ld ÷, doublenegative cells (60). A TUMOR MODEL DIFFERENTIATION

OF THYMOCYTE

+, Recently, independentexperimental evidence was obtained that J1 ld doublenegative, T cell receptor negative lymphob|astsare the immediate precursorsof J1 ld÷, doublepositive, T cell receptor positive cells, which then mayserve as immediateprecursorsfor single positive, T cell receptor positive cells. A double negative, J1 ld +, CD3-AKRT cell thymoma was found whichcontained fl and ), rearrangements. Uponintrathymic injection, double negative clones that had a stable phenotypein vitro ÷, CD8 ÷, CD3 ÷+ changed and produced progeny of the CD4 and ~tfl phenotypein vivo. The double positive progenyexhibited identical fl

Annual Reviews

Annu. Rev. Immunol. 1988.6:309-326. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

T LYMPHOCYTE

DEVELOPMENT

319

and ~ rearrangementsas the doublenegative cells emphasizingthe clonal relatedness of the two populations. Againthe doublepositive cells could be clonedin vitro andreinjected into the thymus.In this waysingle positive ÷, CD8-cells expressing CD3associated 0t, fl heterodimerscould be CD4 obtained from the injected double positive cells (unpublishedresults). Withthe reservation that this represents a tumormodeland the unlikely possibility that the transition fromdoublepositive to single positive cells is simplydue to a loss of the CD8genes, this modelsuggests precursorproduct relationships of thymocytesubpopulations such that receptor negative J1 ld +, CD4-,CD8-cells are the immediateprecursors of ~, fl ÷, CD8 ÷ cells, someof whichare receptor positive double positive CD4 the immediateprecursorcells of receptor-expressingsingle positive cells. SELECTION OF THYMOCYTES THEIR ANTIGEN SPECIFICITY

ACCORDING

TO

Thepreviously discussed experimentsindicate that T cells acquire intrathymically their antigen receptors as well as their potential to become effector cells after antigenic stimulation. It followsthat the thymusmust be the main organ wheredevelopingT cells adjust to self-antigens and learn self-nonself discriminations. Experimentaldata are consistent with the viewthat this adjustmentinvolvespositive as well as negativeselection of T cells expressingcertain antigenspecificities. Theevidencefor positive selection is indirect andless compellingthan that for negativeselection. In discussingthis topic I amgoingto entirely concentrateon0t, fl expressing T cells becauseso little is known aboutthe specificity of y, ~ receptors. Experiments and Considerations Consistent with the Positive Selection of Thymocytes by Thymus Epithelial Cells ÷, CD8 ÷ cells ofCD3 ÷ as well as CD3The fact that double positive CD4 +, CD8 + cells are phenotype die in the thymus mayindicate that CD4 programmed to die unless they express a CD3associated ~t, fl heterodimer that rescues these cells from cell death. This maybe achievedby binding of the receptor to MHC antigens on thymusepithelium. Thusall ~, fl ÷, CD8 ÷ woulddie because they have no receptor, while the negative CD4 ÷, CD8 ÷ cells woulddie becausethey vast majority of 0~, fl positive CD4 express a receptor of inappropriate specificity. A positive selection by MHC antigen on thymusepithelium wouldbe compatiblewith a series of experiments showingthat MHC antigens select maturing thymocytesfor recognition of antigen in association with MHC proteins expressed in the thymus(97-99). Of course, all of these experimentsinvolve in vivo immunizationof otherwise manipulated animals, and it is that area of

Annual Reviews

320 yon BOEHMER

Annu. Rev. Immunol. 1988.6:309-326. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

research on T cell developmentwhichrequires moredirect experimental evidencerefuting or supportingthe aboveinterpretations. Experimental Evidence Supportin# Neyative Selection of Thymocytes Failure to be rescuedfromcell deathis of coursealso negativeselection. This is not the type of negative selection I amdiscussing here. Negative selection discussed here is viewedas the silencing of clones that could becomeautoaggressivebecausetheir receptors bind to self-antigens with sufficiently high affinity to be activated by these. This negativeselection has been namedimmunologicaltolerance, and the debate has continued over manyyears whethersuch tolerance could be achievedby the deletion of autoreactive T cell clones. Veryrecent experimentsare consistent with the notion of clonal deletion. Oneparticular V~17gene segmentendowed T cells with specificity for MHC encodedIE proteins irrespective of the D~,Ja, V~andJ~ segmentsexpressedby these T cells. Cells expressingthis particular Va segmentmakeup several percent in IE negative animals wherethey can be detected by appropriate antibody. Theyoccur in very low numbersin animalsexpressingIE proteins. In IE positive animalsthe doublepositive cortical but not the single positive medullarycells expressed the V~17segment.It wasarguedtherefore that deletion of the V~17expressing occurred somewhereduring the transition from double positive to single positive cells. If in the IE positive animal the Va17determinant recognizedby the Va~7antibody was not simply masked,this experiment indicates that clonal deletion is one physiologicalmechanism of achieving immunologicaltolerance (100, 101). It maynot be the only mechanism, but it is the first to be definedwith someprecision. Thymus grafting experimentssuggest that thymusepithelial cells cannot induceclonal deletion since micegrafted with allogeneicthymicepithelial cells contain T cells with receptors specific for allogeneic MHC antigens of the graft donor(102, 103). Thus,clonal deletion appearsto require the expressionof tolerogens on hemopoieticcells. Oneinteresting aspect of these grafting experimentsis that micegrafted with epithelial cells are unable to reject subsequentgrafts from the samedonor, i.e. a form of tolerance is observedwhereprecursor cells can be activated in vitro but cannotbe inducedto effector function in vivo. Themechanism of this form of tolerance remainsto be determined. A HYPOTHETICAL DEVELOPMENT

SCHEME OF T CELL

The aboveinformation maybe incorporated in a schemeof T cell developmentwhichat this time appears consistent with mostof the experimental

Annual Reviews T LYMPHOCYTE

DEVELOPMENT

321

data. Direct evidence, however,is often missing, and future experiments maydisprove someor all of the scheme.

Annu. Rev. Immunol. 1988.6:309-326. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Gene Rearran#ementin Early Thymocytes Hemopoietic cells enter the thymusprior to rearrangementof g,/3, and 7 genes and begin rearrangement.The first loci to undergorearrangement are ~ and 7 and probably6. Productive 7 and 6 rearrangementsresult in a CD3associated 76 heterodimer,and these cells are the first functional cells to be formedin the thymus.If fl rearrangement is successfulbefore 7 rearrangement,this signals the cell to rearrange g genesegments.Thus, dependingon whichloci productive rearrangementsoccur first, the cell will endup as an g, fl or 7, 6 expressingcell. The7, 6 cells are not considered further in this discussion. Transition

from Double Negative to Double Positive

Cells

The ~z gene segmentrearranging cells becomedoublepositive cells and, dependingon whetherthe ~ rearrangementsare productive, becomeCD3+ + double positive cells are or CD3-double positive cells. Thusthe CD3 the first cells to expressthe ~,/3 T cell receptorfor antigenduringontogeny. Positive Selection and Determination of the Single Positive Phenotype +, CD8 ÷ cells and most of the CD3 ÷, CD4 ÷, + All of the CD3-, CD4 CD8 ÷, CD4 ÷, CD8 ÷ cells whose~,/3 cells die intrathymically. Thosefew CD3 receptor binds to MHC antigen on epithelial cells are rescued from cell death. Depending on whetherthe ~, // receptor binds to class I or class II MHC antigen, the expression of either CD4or CD8is suppressed. If the receptor binds by chance to both class I and class II MHC antigen, both CD4and CD8expression are suppressed, and the cells becomedouble negative,J1 ld-, ~, // receptorexpressingcells whichappearrelatively late in ontogeny.

Negative Selection and Functional Maturation Thepositively selected cells are screenedby hemopoieticcells, and those expressingsufficiently high affinity for self antigens are eliminated.After this eventthe selected single positive cells acquirefunctional maturityand are ready to leave the thymus.It mustbe assumedthat by somemechanism the positive selection requires less affinity of the g,/~ receptorsthan the negative selection becauseotherwiseall positively selected cells would subsequentlybe eliminatedby the negativeselection (47). I consider the above modelconclusively disproven if it can be shown that the functional single positive thymoeytes belongto a different lineage than the nonfunctionaldouble positive thymocytes.

Annual Reviews 322 vor~

BOEI-IMER

Annu. Rev. Immunol. 1988.6:309-326. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Literature Cited 1. Cold Spring Harbor Symposia on Quantitative Biology. 1977. Vol. 19. Cold Spring Harbor, Me. 2. Miiller, G., ed. 1981. T-cell clones. Immunol.Rev. 54:27 3. Haskins, K., Kubo, R., White, J., Pigeon, M., Kappler, J., Marraek,P. 1983. The major histocompatibifity complex-restrictedantigen receptor on T cells. I. Isolation witha monoclonal antibody. J. Exp. Med.157:1149 4. Meuer,S. C., Hodgdon,J. C., Hnssey, R. E., Protentis, J. P., Schlossman, S. F., Reinherz, E. L. 1983. Antigenfike effects of monoelonalantibodies directed at receptors on humanT cell clones. J. Exp. Med.158:988 5. Hedrick,S. M., Cohen,D. I., Nielsen, E. A., Davis, M.M. 1984. Isolation of eDNA clones encodingT cell-specific membrane-associated proteins. Nature 308:149 6. Yanagi,Y., Yoshikai,Y., Leggett, K., Clark, S. P., Aleksander,I., Mak,T. W. 1984. A humanT cell-specific eDNA clone encodesa protein having extensive homology to immunoglobulin chains. Nature308:145 7. Chien,Y., Becker,D. M., Lindsten,T., Okamura,M., Cohen,D. I., Davis, M. -M.1984. A third type of murineT-cell receptor gene. Nature312:31 8. Saito, H., Kranz, D. M., Takagaki, Y., Hayday,A. C., Eisen, H. N., Tonegawa, S. 1984. A third rearranged and expressedgenein a clone of eytotoxie T lymphoeytes.Nature 312:36 9. Dembir, Z., Haas, W., Weiss, S., McCubrey, J., Kiefer, H., yon Boehmer, H., Steiumetz, M. 1986. Transferof specificity by murinect and /3 T-cell receptor genes.Nature320:232 10. von Boehmer,H., Haas, W., eds. 1985. T cell clones. Amsterdam: Elsevier 11. Raft, M. C. 1971. Surface antigenic markers for distinguishing T and B lymphoeytesin mice. Transpl. Rev. 6: 52 12.Samelson, L. E., Harford, J. B., Klausner,R. D. 1985.Identification of the componentsof the routine T cell antigen receptor complex.Cell 43:223 13. Brenner, M. B., Trowbridge, I. S., Strominger,J. L. 1985. Cross-linking of humanT cell receptor proteins: Associationbetweenthe T cell idiotype fl subunit and the T3 glyeoprotein heavysubunit. Cell 40:183 14. Leo, O., Foo, M., Sachs, D. H., Samelson, L. E., Bluestone,J. A.1987.Identification of a monoclonalantibody

specific for a murineT3polypeptide. Proc. NatL Acad. Sci. USA84:1374 15. Williams,A. F., Galfrr, F. G., Milstein, C. 1977. Analysesof cell surfaces by xenogeneic myeloma hybrid antibodies: Differentiation antigensof rat lymphoeytes.Cell 12:663 16. Dialynas,D. P., Quan,Z. S., Wall,K. A., Pierres, A., Quinthns,J., Loken, M.R., Pierres, M., Fitch, F. W.1983. Characterization of the murineantigenic determinant, designated L3T3a, recognized by monoclonal antibody GKI.5: Similarity of L3T4 to the humanLeu3/T4molecule. J. Imrnunol. 131:2445 17. Ledbetter, J. A., Herzenberg, L. A. 1979. Xenogeneicmonoclonal antibodies to mouse lymphoid differentiation antigens. Immunol.Rev. 47: 63 18. Kisielow, P., Hirst, J., Shiku, H., Beverley, P., Hoffmann,N. K., Boyse, E., Oettgen, H. 1975. Ly antigens as markersfor functionally distinct subpopulations of thymus derived lymphocytesof the mouse.Nature253:219 19. Zinkernagel, R. M-, Doberty, P. C. 1979.MHC-restricted cytotoxic T cells: Studies on the biological role of polymorphicmajor transplantation antigens determining T-cell restrictionspecificity, function, and responsiveness. Ado.Irnmunol.27:51 20. Schwartz, R. H. 1985. T-lymphocyte recognition of antigen in association with geneproductsof the majorhist0compatibility complex.Ann. Rev. Imrnunol. 3:237 21. Babbitt, B. P., Allen, P. M., Matsueda, G., Haber, E., Unanue, E. R. 1985. Bindingof immunogenic peptides to Ia histocompatibility molecules. Nature 317:359 22. Buus,S., Sette, A., Colon,S. M., Jenis, D. M., Grey, H. M.1986. Isolation and characterization of antigen-Ia complexes involvedin T cell recognition. Ce1147:1071 23. Steinmetz, M., Hood, L. 1983. Genes of the major histocompatibility complex in mouseand man. Science 222: 727 24. Townsend,A. R. M., Rothbard, J., Gotch,F. M., Bahadur,G., Wraith, D., MeMichael, A. J. 1986. Theepitopes of influenza nucleoprotein recognizedby cytotoxic T lymphocytescan be defined with short synthetic peptides. Cell 44: 959 25. Maryanski,J. L., Pala, P., Corradin,

Annual Reviews

Annu. Rev. Immunol. 1988.6:309-326. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

T LYMPHOCYTE DEVELOPbIENT 323 C., Jordan,B. R., Cerottini, J.-C. 1986. H-2restricted cytolytic T cells specific for HLAcan recognize a synthetic HLApeptide. Nature 324:578 26. Morgan,D. A., Ruscetti, D. W.,Galls, R. C. 1976. Selective in vitro growth of T lymphocytes from normal human bone marrow.Science 193:1007 27. Smith, K. A. 1980. T-cell growth factor. Immunol.Reo. 51:337 28. Howard,M., Farrar, J., Hilfiker, M., Johnson, B., Takatsu, K., Hamaoka, T., Paul, W.E. 1982. Identification of a T cell-derived B cell growthfactor distinct from interleukin 2. J. Exp. Med. 155:914 29. Noma,Y., Sideras, P., Naito, T., Bergstedt-Lindquist, S., Azuma,C., Severinson, E., Tanabe,T., Kinashi,T., Matsuda, F., Yaoita, Y., Honjo,T. 1986. Cloning of cDNA encoding the murine IgGl inductionfactor by a novelstrategy using SP6 promoter. Nature 319: 30. Klein, R. J., Raulet,D. H., Pasternack, M.S., Bevan,M. J. 1982. CytotoxicT lymphocytes produce immuneinterferonin responseto antigenor mitogen. J. Exp. Med. 155:1198 31. Kanemori,H., Kondo,S., Kinoshita, M., Suzuki, N., Saito, Y., Sabo, H., Matsunami,N., Ishida, N., Skimizu, A., Honjo,T. 1986. Intedeukin2 receptor: Structure, functionandexpression. ColdSprint? HarborSyrup. Quant.BioL 57:739 32. Borst, J., van de Griend, R. J., van Oostveen,J. W., Ang,S.-L., Melief, C. J., Seidman,J. G., Bolhuis, R. L. H. 1987. A T-cell receptor v/CD3complex found on cloned functional lymphocytes. Nature325:683 33. Moingeon, P., Jitsukawa,S., Faure,F., Troalen,F., Triebel, F., Graziani,M., Forestier, F., Bellet, D., Bohuon,C., Hercend,T. 1987. A v-chain complex formsa functional receptor on cloned human lymphocytes with natural killer-like activity. Nature325:723 34. von Boehmer,H., Haas W.1981. H-2 restricted cytolytic and noncytolyticT cell clones: Isolation, specificity and functional analysis. ImmunoL Rev. 54: 27 35. Ferrini, S., Bottino, C., Biassoni,R., Poggi, A., Sekaly,R. P., Moretta,L., Moretta, A. 1987. Characterization of CD3+,CD4-, CD8-clones expressing the putativeT cell receptorV geneproduct. J. Exp. Med.166:277 36. Yagiie, J., White,J., Coleclough,C., Kappler, J., Palmer, E., Marrack,P. 1985.TheT cell receptor: The¯ and fl

37.

38.

39.

40.

41.

42.

43.

44.

45. 46.

47. 48.

chainsdefine idiotype, and antigen and MHC specificity. Cell 42:81 Dembi6,Z., von Boehmer,H., Steinmetz,M.1986.T cell receptor a andfl genes are necessary and su~icient for MHC restricted antigen recognition. ImmunoLToday 7:308 Saito, T., Weiss, A., Miller, J., Norcross,M. A., Germain,R. N. 1987. Specificantigen-Iaactivation of transfected humanT cells expressingmurine Ti ~tfl-humanT3 receptor complexes. Nature 325:125 Gebert, J., Couglet,C., Zamoyska, R., Parues, J. R., Sehmitt-Verhulst, A., Malissen, B. 1987. Reconstitution of MHC class I specificity by T-cell receptor andby F-2 genetransfer. Cell 50: 545 Swain,S. L., Dialynas, D. P., Fitch, F. W., English, M. 1984. Monoclonal antibody to L3T4blocks the function of T cells specific for class II major histocompatibifitycomplexantigens. J. ImmunoL132:1118 MacDonald, H. R., Glasebrook,A. L., Bron, C., Kelso, A., Cerottiui, J.-C. 1982.Clonalheterogeneityin the functional requirement for Lyt-2/3 molecules on eytolytie T lymphoeytes (CTL):Possible implications for the affinity of CTLantigen receptors. lmmunoLRev. 68:89 Hfinig, T. 1984. Monoclonalanti-Lyt2.2 antibody blocks lectin-dependent cellular cytotoxicity of H-2-negative target cells. J. Exp. Med.159:551 Wassmer,P., Chart, C., L6gdberg,L., Shevach,E. M.1985. Roleof the L3T4antigen in T cell activation. II. Inhibition of T cell activation by monoclonal anti-L3T4 antibodies in the absenceof accessorycells. J. lmmunoL 135:2237 Dembi6, Z., Haas, W., Zamoyska, R., Parries, J., Steinmetz, M., von Boelmaer,H. 1987. Transfectionof the CD8gene enhancesT-cell recognition. Nature 326:510 Swain,S. L. 1983. T cell subsets and the recognition of MHC class. Immunol.Rev. 74:129 Goverman,J., Hunkapillar, T., Hood, L. 1986.A speculativeviewof the multicomponentnature of T cell antigen recognition. Cell 45:475 yon Boehmer,H. 1986. The selection of the ~t, fl heterodimeric T-cellreceptor for antigen. Irnmunol.Today7:333 Emmrich,F., Strittmatter, V., Eichmann,K. 1986. Synergismin the activation of human CD8T cells by crosslinkingthe T cell receptor complex

Annual Reviews

Annu. Rev. Immunol. 1988.6:309-326. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

324 vor4 BOEHMER with the CD8differentiation antigen. Proc. Natl. Acad. Sci. USA83:8298 49. Brenner,M. B., McLean,J., Dialynas, D. P., Strominger,J. L., Smith,J. A., Owen,F. L., Seidman,J. G., Ip, S., Rosen,F., Krangel,M.S. 1986.Identification of a putative second T-cell receptor. Nature322:145 50. Lew, A. M., Pardoll, D. M., Maloy, W.E., Fowlkes,B. J., Kruisbeek,A., Chew,S. F., Germain, R. N., Bluestone, J. A., Schwartz,R. H., Coligan, J. E. 1986. Characterizationof T cell receptor ~,-chain expressionin a subset of murine thymocytes. Science 234: 1410 51. Saito, H., Kranz, D. M., Takagaki, Y., Hayday,A. C., Eisen, H. N., Tonegawa,S. 1984.Completeprimarystructure of a heterodimericT-cell receptor deduced from cDNAsequences. Nature 309:757 52. German,R. D., Doherty,P. J., Raulet, D. H. 1986. Diversity, rearrangement andexpressionof murineT cell 2: genes. Cell 45:733 53. Chien, Y., Iwashima,M., Kaplan, K. B., Elliott, J. F., Davis,M.M.1987.A newT-cell receptor genelocated within the alpha locus and expressedearly in T-cell differentiation. Nature327:677 54. Davidson,W.F., Dumont,F. J., Bedigian, H. G., Fowlkes,B. J., Morse,H. C. 1986. Phenotypic, functional and moleculargenetic comparisonsof the abnormal lymphoid cells of C3Hlpr/Ipr andC3H-gld/gldmice. J. Immunol. 136:407 55. Roths,J. B., Murphy,E. D., Eicher, E. M. 1984. A newmutation, gld, that produceslymphoproliferationand autoimmunity in C3H/HeJmice. J. Exp. Med. 159:1 56. Haskins, K., Hannum,C., White, J., Roehm,N., Kubo, R., Kappler, J., Marrack,P. 1984.Theantigen-specific, major histocompatibility complexrestricted receptor on T cells. VI. An antibodyto a receptor allotype. J. Exp. Med. 160:452 57. Sim,G. K., Augustin, A. A. 1985. V/~ gene polymorphismand a major polyclonal T cell receptoridiotype.Cell 42: 89 58. Bruce, J., Symington,F. W., McKearn, T. J., Sprent, J. 1981. A monoclonal antibody discriminating betweensubsets of T and B cells. J. Immunol.127: 2496 I. S., Lesley, J., Schulte, 59. Trowbridge, R., Hyman,R., Trotter, J. 1982. Biochemicalcharacterization and cellular distribution of a polymorphicmurine

cell surface glycoproteinexpressedon ]ymphoidtissues, lmmunogenetics15: 299 60. Bluestone,J. A., Pardoll, D. M., Sharrow, S. O., Fowlkes,B. J. 1987.Characterization ofmurinethymocyteswith CD3associated T cell receptor structures. Nature326:87 61. Crispe, I. N., Moore, M. W., Husmann,L. A., Smith, L., Bevan,M. J., Shimenkavitz, R. P. 1987. Differentiation potential of subsets of CD4-, CDS-thymocytes. Nature 329:336 62. Sc611ay, R., Chen, W.-F., Shortman, K. 1984.Thefunctional capabilities of cells leaving the thymus.J. Immunol. 132:25 63. Kisielow, P., Leiserson, W., von Boehmer,H. 1984. Differentiation of thymocytes in fetal organ culture: analysis of phenotypic changes accompanyingthe appearance of cytolytic andinterleukin 2-producingcells. J. Immunol. 133:1117 64. Rothenberg, E., Lugo, J. P. 1985. Differentiationandcell divisionin the mammalianthymus. Devel. Biol. 112: 1 65. Raulet, D. H. 1985. Expression and function of interleukin-2 receptors on immaturethymocytes. Nature 314:101 66. Ceredig, R., Lowenthal, J, W., Nabholz,M., MacDonald,H. R. 1985. Expressionofinterleukin-2receptorsas a differentiation markeron intrathymic stemcells. Nature314:98 67. yon Boehmer, H., Crisanti, A., Kisielow, P., Haas, W.1985. Absence of growthby most receptor-expressing fetal thymocytesin the presence of interleukin-2. Nature314:539 68. Palacios, R., von Boehrner,H. 1986. Requirementsfor growth of immature thymocytesfrom fetal and adult mice in vitro. Eur. J. Immunol.16:12 S. N., Sailor, R. 69. Lugo,J. P., Krishman, D., Rothenberg, E. 1986. Early precursor thymocytescan produceinterleukin 2 uponstimulation with calcium ionophore and phorbol ester. Proc. Natl. Acad. Sci. USA83:1862 70. Palacios, R., Sideras, P., von Boehmer, H. 1987. Recombinant interleukin 4/BSF-I promotesgrowth and differentiation of intrathymic T cell precursors from fetal micein vitro. EMBO J. 6:91 71. Jenkinson, E. J., Kingston,R., Owen, J. J. T. 1987,IL-2receptorsplaya functional role in intrathymicT-cell precursor proliferation andthe generation of cells expressing T cell receptors. Nature 329:160

Annual Reviews

Annu. Rev. Immunol. 1988.6:309-326. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

T LYMPHOCYTEDEVELOPMENT 72. Skinner, M., LeGros, G., Marbrook, J., Watson, J. D. 1987. Development of fetal thymocytesin organ cultures. Effect of interleukin-2. J. Exp. Med. 156:1481 73. Lowenthal, J. W., Howe, R. C., Ceredig, R., MacDonald,H. R. 1986. Functional status of interleukin 2 receptors expressedby immatureLy2-, L3T4-thymocytes. J. Immunol. 137: 2579 R., Schulte,R. 1985. 74. Lesley,J., Hyman, Evidencethat the Pgp-1glycoproteinis expressed on thymus-homing progenitor cells of the thymus.Cell. Immunol. 91:397 R. 1985. 75. Lesley,J., Trotter, J., Hyman, ThePgp-1antigen is expressedon early fetal thymocytes.Immunogenetics 22: 149 76. Scollay, R. G., Butcher,E. C., Weissmann,I. L. 1980. Thymuscell migration: Quantitativeaspects of cellular traffic from the thymusto the periphery in mice. Eur. J. Immunol.10:210 77. Reichert, R. A., Gallatin, W. M., Butcher,E. C., Weissmann, I. L. 1984. A homingreceptor-bearing cortical thymocytesubset: Implications for thymuscell migration and the nature of cortisone-resistant thymoeytes. Cell 38:89 78. Shortman,K., Wilson, A., van Ewijk, W., Scollay, R. 1987. Phenotypeand localization of thymocytesexpressing the homingreceptor-associatedantigen MEL-14:Argumentsfor the view that most maturethymocytesare located in the medulla. J. Immunol.138:342 79. Stutman, O. 1978. Intrathymic and extrathymic T cell maturation, lmmunol. Rev. 42:138 80. Piquet, P.-F., Irle, E., Kollatte, E., Vasalli, P. 1981. Post-thymicT lymphocyte maturation during ontogenesis. J. Exp. Med.154:581 81. Matsuyama,M., Widrowski, M. N., Metcalf, D. 1966. Autoradiographic analysis of lymphopoiesis and lymphocytemigrationin micebearingmultiple thymusgrafts. J. Exp. Med.123: 559 82. Shortman,K., Jackson, H. 1974. The differentiation of T lymphocytes.I. Proliferation kinetics and interrelationships of subpopulations of mousethymuscells. Cell. Immunol.12: 230 83. McPhee,D., Pye, J., Shortman, K. 1979. The differentiation of T lymphocytes. V. Evidencefor intrathymic death of most thymocytes. ThymusI: 151

325

84. yon Boehmer,H., Snodgrass,R., Kisielow, P. 1986. T cell developmentand tolerance. In Mechanismsof Host Resistance for Infectious Agents, Tumorsand Allografts, ed. A. North, R. Steinmann, p. 38. NewYork: Rockefeller 85. Crispe, I. N., Bevan, M. J. 1987. Expressionand functional significance of the Jlld marker on mouse thymocytes.J. Immunol.138:2013 86. Fowlkes,B. J., Edison,L., Mathieson, B. J., Chused,T. M.1985.Early T lymphocytes. J. Exp. Med.162:802 87. Ezine, S., Weissman, I. L., Rouse,R. V. 1984.Bonemarrow cells give rise to distinct cell cloneswithinthe thymus. Nature 309:629 88. Kingston,R., Jenkinson, E. J., Owen, J. J. T. 1985. A single stemcell can recolonize an embryonicthymus,producingphenotypically distinct T-cell populations. Nature317:811 + murine T cells 89. Smith, L. 1987. CD4 + precursors developfrom CD8 in vivo. Nature 326:798 90. Cristanti, A., Colantoni, A., Shodgrass, R., von Boehmer, H. 1986. Expressionof T cell receptors by thymocytes: in situ staining and biochemical analysis. EMBO J. 5:2837 91. Farr, A. G., Anderson,S. K., Marrack, P., Kappler,J. 1985.Expression of antigen-specific, MHC-restricted receptors by cortical and medullarythymocytes in situ. Cell 43:543 92. Snodgrass,H. R., Kisielow,P., Kiefer, M., Steinmetz, M., von Boehmer,H. 1985. Ontogenyof the T-cell antigen receptor within the thymus. Nature 313:592 93. Raulet, D. H., Garman,R. D., Saito, H., Tonegawa,S. 1985. Developmental regulation of T-cell receptor gene expression. Nature314:103 94. Snodgrass, H. R., Dembir,Z., Steinmetz, M., von Boehmer, H. 1985. Expressionof T-cell antigen receptor genes during fetal developmentin the thymus. Nature 315:232 95. Williams, G. T., Kingston, R., Owen, M.J., Jenkinson,E. J., Owen,J. J. T. 1986. A single micromanipulatedstem cell givesrise to multipleT-cell receptor gene rearrangementsin the thymusin vitro. Nature324:63 96. Wilson,A., Ewing,T., Owens,T., Scollay, R., Shortman, K. 1981.T cell antigen receptor expression by subsets of early Ly3 , L3T4 thymocytes. J. Immunol.In press 97. Bevan, M. J. 1977. In a radiation chimaera,host H-2antigens determine

Annual Reviews

Annu. Rev. Immunol. 1988.6:309-326. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

326

VON BOEHMER

immuneresponsivenessof donor cytotoxic cells. Nature269:417 98. Zinkernagel, R. M., Callahan, G. N., Althage,A., Cooper,S., Klein, P. A., Klein, J. 1978. Onthe thymusin the differentiation of "H-2 self-recognition" by T cells: Evidencefor dual recognition? J. Exp. Med.147:882 99. von Boehmer,H., Haas, W., Jerne, N. K. 1978. Major histocompatibility complex-linked immune-responsiveness is acquiredby lymphocytes of lowrespondermicedifferentiating in thymus of high-responder mice. Proc. Natl. Acad. Sci. USA75:2439 100. Kappler, J. W., Wade,T., White, J., Kushnir, E., Blackman,M., Bill, J., Roehm,N., Marrack,P. 1987. A T cell

receptor Vfl segmentthat impartsreactivity to a class II major histocompatibility complexproduct. Cell 49:263 I01. Kappler, J. W., Roehrn,N., Marrack, P. 1987.T cell toleranceby clonalelimination in the thymus.Cell 49:273 102. Jenkinson,E. J., Jhittay, P., Kingston, R., Owen,J. J. T. 1985.Studiesof the role of the thymicenvironmentin the induction of tolerance to MHC antigens. Transplantation39:331 103. von Boehmer,H., Sehubiger, R. 1984. Thymocytesappear to ignore class I majorhistocompatibilitycomplexantigens expressed on thymusepithelial cells. Eur. J. ImmunoL 14:1048

Annu. Rev. Immunol. 1988.6:309-326. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Annu. Rev. Immunol. 1988.6:309-326. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Annual Reviews www.annualreviews.org/aronline

Annu. Rev. Immunol. 1988.6:327-358. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Ann.Rev. Immunol.1988. 6: 327-58 Copyright© 1988by AnnualReviewsInc. All rights reserved

V GENES ENCODING AUTOANTIBODIES: MOLECULAR AND PHENOTYPIC CHARACTERISTICS Constantin

A. Bona

MountSinai School of Medicine, Departmentof Microbiology, One Gustave L. Levy Place, NewYork, NY10029 INTRODUCTION The immunerepertoire generated by rearrangement of discrete DNA segments,the genes encodingthe specificity of lymphocytereceptors, is composedof clones able to recognize both foreign and self antigens. Starting with Paul Ehrlich, immunologistsheld for a long time that autoreactive clones are harmful(horror autotoxieus). Indeed, strong evidence suggeststhat someautoantibodieshavedeleterious effects and are responsible for pathogenictissue-damagingreactions in autoimmune diseases. Whenantibodies from patients with autoimmunediseases (autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, myasthenia gravis, autoimmune thyroiditis, type I diabetes, pemphigusvulgaris, myelomapolyneuropathy)are injected into animals, they induce symptoms observedin the correspondinghumandiseases. To explain tolerance to self antigens, Burnet(1) proposedthat autoreactive clones are deleted during ontogeny.Enthusiasmfor this explanation wanedduring the last decadesdue to findings demonstratingthat precursorsof autoreactiveclones are present in normalindividuals in both B and T cell compartments.Surveysof healthy humanpopulations showed the appearanceof autoantibodies, especially with aging (2), and cells binding to labeled self-antigens were detected in the blood of healthy humansubjects (3). Similarly, subsequentto stimulation with B cell polyclonal activators, hybridomasproducing autoantibodies can be easily 327 0732-0582/88/0410~327502.00

Annual Reviews

Annu. Rev. Immunol. 1988.6:327-358. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

328

BONA

obtained both from animals either prone or not prone to autoimmune diseases (4). Nossal et al (5, 6) amendedthe clonal deletion theory adding two variants: "clonal abortion" suggested that the autoreactive clones are aborted in their ability to differentiate and mature, or are "anergic" since autoantigensrepresent a negativesignal. However,these hypothesesdo not explain the occurrenceof rheumatoid factors in healthyindividualsafter vaccinations(7), infectiousdiseases(8), or after immunizationof inbred strains not prone to autoimmune diseases (9, 10). Furthermore,recent studies showedthe ubiquityof B cells secreting autoantibodies (11). Grabar(12) proposedthat ubiquitous natural autoantibodieshave, in fact, a physiologicalrole as carriers of metabolitesor tissue degradation products. Kansari & Fudenberg(13) demonstratedthat autoantibodiescontribute to the clearing of agedcells and, therefore, to the maintenanceof cellular homeostasis. Therefore, two other mechanisms were implicated in self-tolerance. One involves suppressorT cells. Numerous reports indicate alteration of the T helper :T suppressor ratio in autoimmunediseases (14). However, evidencesupportsthe conceptthat the break of self-tolerance is causedby loss of suppressor T cells (15). The secondmechanism invokes breakdown of idiotype regulation. Themajorpattern of idiotype interactions in the steadystate is the suppressionof clonesof B cells specific for conventional antigens(Ab~clones)by clones of antiidiotype(anti-Id) antibodiesor idiotypic specific suppressorT cells (AbEclones). Antigenupsets this equilibrium,and Ab~clones stimulated by antigen escape the suppressiveeffect of Ab2clones (16). However, there is little convincingdata that expansion of autoreaetiveclones is driven by autoantigens. Twomajor features emerge from the studies of humanand animal autoimmune disease. First, the onset of autoimmune diseases is generally agerelated. However, a high variability in the age of onset of auto hemolytic anemia,lupus syndrome,and occurrenceof rheumatoidfactors is observed even in inbred strains such as NZB,B × SB, or 129/Svmice, respectively. Secondly,the importanceof genetic factors in the occurrence of autoimmunedisease was noted in humansas well as in animals. However,in humansubjects a 50%discordance in acquisition of autoimmune disease has been observedin monozygotictwins (15), indicating that nongenetic factors can contribute to the breakingof tolerance. Burnet proposedthat somatic mutations play an important role in the generationof autoreactive("forbidden")clones. This conceptis supported by an observation reported by Diamond& Scharff (17), whoshowedthat a mutant selected amongthe phosphocholine-bindingprotein S 107 bound to DNA.Sequenceanalysis of this protein showeda single point mutation in position 35 of the Hchain, whichapparentlysuffices to alter drastically

Annual Reviews

Annu. Rev. Immunol. 1988.6:327-358. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

V GENES OF AUTOANTIBODIES

329

the specificity of an anti-PC antibody to becomeself-reactive. This predicts that while the production of ubiquitous autoantibodies is physiological, the pathogenic antibodies should be restricted to a clone or a few clones that underwent a mutational event(s). Monoclonal antibodies have been detected by isoelectrofocusing in a fraction of patients with autothyroiditis (18). If the pathogenic autoantibodies are highly restricted, they might generated by precise combinations of VH-VLgene products. Takentogether, these findings suggest that studies of the characteristics of V genes encoding autoreactive antibodies might provide clues for the further understanding of the mechanismsof autoimmunedisease. In particular, elucidation of their idiotypes, phenotypic antigenic markers of these genes, might help to determine whether or not they function as targets of idiotype-determined regulatory processes. Studies of reactivity of B cells of animals prone to autoimmunediseases showedthat they do not exhibit a generalized defect in tolerance against various antigens. A single study described a tolerance defect for TNP antigens in continuous B cell lines obtained from an autoimmunestrain (19). The ability to synthesize pathogenic autoantibodies might be related to a specific dysfunction which lies in B cells. Hypergammaglobulinemia, a hallmark of numerous autoimmunediseases (20), and acceleration maturation of B cells in autoimmunestrains (10, 21) also argue for intrinsic defect of B cells.

CHARACTERISTICS OF REARRANGED V GENES OF AUTOANTIBODIES AND GERMLINE GENES OF AUTOIMMUNE STRAINS Molecular studies aimed at investigating the characteristics of V genes encoding autoantibodies were designed to address several questions related to the organization of genes, utilization of various V gene families, and the polymorphismof these genes in strains prone to autoimmunediseases.

Organization and Structure of V Genes Encodin9 Autoantibodies Diversity of autoantibodies, like that of antibodies specific for foreign antigens, is generated as a result of rearrangements of DNAsegments encoding the variable regions of the heavy and light chain (V, D, J for heavy chain and V, J for light chain). Protein sequence of variable regions of two types of humanautoantibodies (cold hemagglutinins and rheumatoid factors) and nucleotide sequences of V genes of murine autoantibodies with various specificities together clearly showedthat the coding sequence of V genes of autoanti-

Annual Reviews

Annu. Rev. Immunol. 1988.6:327-358. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

330

BONA

bodiesdoesnot differ fromthose of antibodiesspecific for foreignantigens in a general way. Studyof N-terminalprotein sequencesof kappalight chains of 9 human cold agglutinins showedthat they belong to V~:III subgroupswhichare actually the most prevalent subgroupsin normalserum(VI(III is borne 11%of humanserum immunoglobulins). Sequencing of these proteins showedseveral substitutions comparedto the prototype sequence (22). Protein sequencingof humanmonoclonalproteins exhibiting rheumatoid factor (RF)activity also showedthat they used restricted subgroups:RFs expressingthe Wacross-reactive idiotype use VHIandV~:IIIb, while those expressing Po IdX used VHIand V~:IIa. In spite of numerousaminoacid differences, these RFshave highly homologoussequences in the CDRII andCDRIII regionsof V~:(23, 24), andantibodiesspecific for the synthetic peptides correspondingto CDRIIand CDRIIIreact with separated light chains(25). Nucleotide sequence of a humanV~:III germline gene (Humk305), recently cloned fromhumanfetal liver (26), showedthat its deducedamino acid sequencediffers at four residues from that proposedas the V~IIIb prototype. Aprobecontaining this germlinegenein Southernblots detects the rearranged Vr: genes of humanhybridomasproducing RFsthat share a cross-reactive idiotype related to the CDRIIof the V~:IIIb light chain. Whilelittle homologywas observedin the sequences of the VHdomains of RFs expressing WaIdX, a high degree of homologywas noted in the VM regions of RFsexpressingPo IdX(27), suggestingthat the Vr~can also play a role in the self-reactivity of RFs. Analysisof VMand Vr: used in murine RFs by Northern blotting or nucleotide sequencing showedthat while the VHgenes used are quite different from one another, there are only a few Vr~subgroupsused (28, 29) (Table1). Comparison of nucleotide sequencesof VI~genesexpressedin a large panel of RFsshowedthat there are few or no somatic mutations. Indeed, very few somaticmutations have been observedin the sequenceof six monoclonalantibodies specific for bromelain-treated mousered blood cells obtained from nonimmunized NZBor CBA/Jmice. The VKgenes were almost identical, and the Vns differedbyonly six substitutionswhichmightreflect allelic differences(30). VHsequencesof these antibodies showvery little homology with VHgenes identified so far. By contrast, Vns of four RFs from WaIdX+ groups showed high homology with the VHof a humananticytomegalovirus monoclonalantibody (31, 32). Sequencesof four monoclonalantibodies from lupus-prone mice exhibiting various specificities (DNA,histone, and rheumatoidfactors) showed that, while all used Vngenesfromthe VHJ558family, they used different VKgenes. Important nucleotide differences amongthe V~genes (19.3-

Annual Reviews V GENES OF AUTOANTIBODIES

331

Table 1 Representation of V gene famialies amongmurine RFs

Annu. Rev. Immunol. 1988.6:327-358. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

VK 36-09 X24 36-60 J606 J558 S107 QPC52 7183

1/57 1/57 6/57" 2/57 27/57 0/57 3/57 17/57

1 4 8 9 10 19 21 23 24

8/36 4/36 11/36 0/36 0/36 10/36 0/36 1/36 2/11

~ Thedata presentedin this table representa summaryof findings reported by Schlomchiket al (28, 29), Manheimer-Lory et al (35), Painter al (53), and*Arantet al (39). Va and VKfamilies were determined by sequenceof eDNA (28, 29, 35), Northernblotting (35, 53) or slot blotting(35, 39).

27.8%)suggest that these autoantibodies used H chains derived from different germlinegenesof the VHJ558family(33). Oneof these V. genes exhibited high homologyto a Vr~ germline gene from which anti-NP antibodiesare derived(34). A study of nucleotide sequencesof the entire rearranged Vr~ genes of two hybridomas producing RFs, one from a BALB/cmouseimmunized with Y. enterocolitica and another from a 129/Sv mouse,a memberof a strain prone to produceRFswas carried out. The study showedno major differences in the organization of V genessince the length of the leader sequence and that of the intron located between leader and coding sequences are very similar to those observed in other Vr~ genes. The sequence of one of these RF V. genes was highly homologousto V. germlinegene from whichthe H chain of anti-NP antibodies are derived; the other useda genefromthe Vr~7183family(35). Theseobservations suggest that the organization of V genes of autoantibodiesis not uniquebut rather is similar to that of antibodiesspecific for foreign antigens. Theyalso indicate that autoantibodies display no restriction in the recombinationof V. and VKwith a particular D or J. or Ji~, respectively.D,Jr~, or Ji~ genesidentified in autoantibodiesinclude examplesof all knowngermline genes. The length of D segmentsvaries between3 and 12, as in antibodies with other specificities. Comparison of sequencesof rearrangedV genes with germlinegenes indicates that the V genes of autoantibodies derive from the samegenepool as do antibodies

Annual Reviews

332

BONA

Annu. Rev. Immunol. 1988.6:327-358. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

specific for foreign antigens, except for those encoding the Br-MRBC specificity. Themoststriking aspect of structural studies, so far, is the paucity of mutations observed in the coding sequences of both VHand VLgenes of autoantibodies. Preferential Utilization of Some V Gene Families in Autoantibodies The murine Vn locus contains between one hundred and several hundred genes; the VKlocus is madeup of two to three hundredgenes. Basedon protein or DNAsequence homology,the genes of Vn and VL loci were classified initially in subgroups(36) and later in families (37). Thus, VHlocus contains 8-10 families (36-09, X24,36-60, J606, J558, S107, QPC52and 7183), whereas the VKlocus contains about 30 subgroups (VK1,..., VK24). It is well knownthat in the case of oligoclonal responseselicited by polysaccharides,a limited set of immunoglobulins makeup the repertoire of antibodies specific for the epitopes of the immunogen. The limitation of the repertoire apparentlyis related to the utilization of genesfromthe samefamily and to restricted VH: VL combinations(38). If autoantibodies are the product of forbidden clones generated by somaticmutationsafter the tolerance step is brokendown,these antibodies could be predictably encodedby a limited pool of Vgenes and restricted to particular VH: VLcombinations.Therefore, it wasimportantto determinethe V gene families used in autoantibodies. This question has been addressed by using various methods--sequencingof cloned rearranged V genes (35) ofcDNAcomplementaryto mRNA extracted from hybridomas (28, 29, 33) or Northernand slot blotting of RNAwith various V probes prototypic for each VHor VI~family(35, 39). Thesemethods,whenapplied to studyingthe Vgenes of RFs, showedthe representation of the majority of VHfamilies, whichwasconsistent with randomutilization of Vngenes fromthe available germlinegenepool with a slight overrepresentationof VH7183. A nonrandomselection of genes from the Vn 36-60 family was only observed in a group of RFs hybridomasobtained from 4-15-weekold MRL/lprmice (39), and of genes from the VH7183 family in hybridomasobtained from 5-6-month-old129/Sv mice (35). In contrast, only a few VKsubgroups have been identified in RFs, namely VK1,VK4,VK8and VK19(Table 1). The combinatorial VH :V L associationis not restricted in RFssince a givenVI~can be associatedwith Vnsfromvarious families. Thus, while only a few Vr: subgroups are used by murine and human RFs, the VHgenes are used randomly.The randomutilization of Vn and

Annual Reviews

Annu. Rev. Immunol. 1988.6:327-358. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

V GENES OF AUTOANTIBODIES

333

several VI¢genefamilies can be related to the nature of putative antigen responsible for the expansionof clones producingRFs, namelyantigenic determinants of globular domains (CH2and CH3)on the Fc fragment. Random utilization of V genefamilies can be related to the heterogeneity of antibody responses elicited by multideterminantantigens. A similar situation wasobservedin the responseagainst multivalent"foreign" antigens such as the hemagglutinin of influenza virus (40, 41). Analternative hypothesis is that RFsare multispecific antibodies producedby clones specific for other antigens that bind to Fc epitopes as a result of crossreactions. This explanation can be supported by observations that human RFsbind to a DNA-histonecomplex(42) or that a murine monoclonal RFbinds to foreign antigens such as GTor GL~(43). Furthermore,wedescribed a categoryof anti-Id antibodies that wehavedesignatedepibodies whichbind idiotypes of RFsas well as Fc fragments(44, 45). Obviously, if the majority of RFsare multispecific antibodies producedby clones recognizinga variety of antigens, then the randomutilization of Vgene familiesandlack of restriction of VI~: VLassociationis not surprising. In this respect, it will be interesting to studyVnandVLfamilies of pathogenic rheumatoidfactors such as those exhibiting cryoglobulin properties and those obtainedfromlymphocytes infiltrating arthritis lesions. Restricted usage of genes from the Vn 36-60 family by RFsobtained from MRL/lprmice (39), whichdevelop an arthritis-like syndrome,and of genes from Vr~ 7183by RFsobtained from old 129/Svmice (35), is indication that pathogenicRFscan be generatedby a limited repertoire. Studies carried out in our laboratory on a large panelof autoantibodies with various specificities originating fromvarious murinestrains suggest a preferential usageof Vr~genesfromfamilies located at the 3’ end of the entire VHregion. The data depicted in Table 2 showthat out of 63 hybridomassecreting autoantibodies, 28 use genesderiving from S107,QPC52 and 7183("3’ families"), 29 fromthe J558family, and only 6 fromthe VHfamilies (36-09, X24, 36-60, and J606). Analysis of the VHSof monoclonalanti-DNA antibodies is in agreementwith our results: 5 of 11 used Vr~J558, one S107,two QPC52,and three 7183VHgene families (46). Furthermore, Bellon et al (4) have shownthat the frequency of hybridomasselected with a Vn7183germlinegeneprobeis significantly higher in NZBand MRL/Ipr mice than in BALB/cmice. NZBand MRL/lpr mice are prone to developingautoimmune diseases. Studyof the binding properties of monoclonalantibodies producedby hybridomasselected with the VH7183 probe showedthat a high proportion exhibit self-binding properties. However,no significant difference in the frequency of autoantibodies amongVH7183+ antibodies has been observed between antibodies derived from normal and autoimmune

Annual Reviews 334

BONA

Tal)le 2 Representation of VHgenefamiliesamong autoantibodies withvariousspecificity

Annu. Rev. Immunol. 1988.6:327-358. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Specificity Thyroglobulin DNA Sm Collagen typeII Intrinsicfactor Microfilaments Myelinbasic protein Thymocytes GBM Br-RBC TSH-R Frequency

Vn36-09 VHX 24 VH36-60VHJ606

1/14

1/5 2/14

VHJ558

4/9 13/23 3/5 5/14 1/1

1/14

VHS107VHQPI252 I/9 1/23 3/23 1/5 2/14

VH718

4/9 6/2~

I/1 3/3 1/1 1/2 1/63

3/63

1/63

1/63

29/63

3/63

7/63

1/2 3/3 1/1 18/6~

strains. This indicates that this family is used for self-reactivity in normal strains as well as in those prone to autoimmunediseases. It is interesting to note that the 3’ VH7183 family is frequently used in early development (47, 48). Therefore, VH7183 gene family usage mayreflect an immature repertoire. The subset of B lymphocytes bearing the Ly 1 marker contains the majority of the precursors of anti-Br-MRBC, anti-thymocyte, and anti-ssDNA antibody-forming cells (49). This subset is expanded in NZB mice (50) and is predominant in motheaten mice (51), a strain developing multiple autoimmunediseases. Lyl B cells have the characteristics of B lymphocytes developing early in postnatal life, expressing high density IgMand little IgD on their surface (52). These results suggested that Lyl B cells might preferentially express 3’ VHgene families. To test this we prepared hybridomas from 1 and 2 month old motheaten (mev) mice to study the representation of VHgenes among those that secrete autoantibodies. For the selection of self-binding antibodies, we used a panel of autoantigens knownto be a target of organspecific antibodies (antibodies specific for myelinbasic protein, intrinsic factor, collagen type II, thyroglobulin, transferrin, acetylcholine receptor, red blood cells, thymocytes, and glomerular basal membrane), and nonorgan specific antibodies (DNA,Sm, Fc fragment, mitochondria, nuclear antigens). Study of the immunochemicalproperties of monoclonal antibodies produced by me° hybridomas showed that the majority of them bind to several autoantigens. This multispecific binding to various autoantigens can be related to the autoimmune syndrome observed in vme characterized by glomerulonephritis, thymic aplasia, anemia, interstitial pneumonitis, and neutrophilic infiltration of skin.

Annual Reviews

Annu. Rev. Immunol. 1988.6:327-358. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

V GENES OF AUTOANTIBODIES

335

Thespecificity of someof these autoantibodiescorrespondsto the autoimmune pathologyof mev mice, such as the antibodies specific for thymocytes, skin, erythrocytes, and glomerularbasal membrane.Other specificities--such as those for myelinbasic protein, thyroglobulin,transferrin, acetylcholine receptor--are not associated with a correspondingpathology (53). Perhapsthe short life time of mev mice (average 61 days) does not allow the manifestation of symptomatology of the disease caused by someorgan-specific antibodies. No knownmechanismsexist for the activation of the precursors of autoantibody-forming cells in motheatenmice. This polyclonal activation maybe related to the production of various lymphokinessuch as B cell maturation factor (54), whichis well documented. Northernblotting analysis of V genes expressed in our small panel of me~ hybridomasproducing autoantibodies showedthat 4 used genes from 5’ VHgene families, 3 from VHJ558, and 8 from3’ VHgenefamilies. Only 5 of 16 autoantibodiesusedgenesfromVI~7183,the most3’. Sincevirtually ÷, our results showthat Lyl.B cells do all B cells of mev miceare LylB not use only the most3’ endVHgenefamilyas wasinitially predicted (49) based on the "young"phenotypecharacterizing this subset. Our results do not exclude potential rearrangement mechanismsthat could conceivablyoccur subsequentto the activation and differentiation of Lyl B cells duringthe two-monthlifetime of motheatenmice. Kleinfeld et al (55) analyzedrearrangement of the productiveallele of a Lyl.Bcell lymphoma and observed the replacementof the initially rearranged Via Q52gene by a VH7183 gene. In contradistinction,a restricted usageof VKfamilies has beenobserved. Thus, among16 hybridomas,4 used VK1,2 VI~4,4 VK10and 2 V~19.Only 4 out of 16 used a VI( fromthe remaining20 V~:families. Since these VI~ gene families are also preferentially used in murinerheumatoidfactors, wehavestudiedthe distribution OfVK|, VI,:4, Vr:8, Vr:10,V~:19,Vi~21,and VI(24amonga larger panel of monoclonal antibodies (Table3). Ourresults showedthat in our panel of 43 autoantibodieswith various specificities, VI(1, V~4,V~:10,and VI~19,highly represented in motheatenautoantibodies, were used by 27 of 43. VI(8, observed to be used in RFs, was identified in 2 of 48. Noneof these autoantibodiesused V~:21and V~24, and10 of 43 used Vr~genesfromthe remainingfamilies of murineautoantibodies. In the human,it wasalso observedthat anti-TSHreceptor autoantibodies in Grave’sdisease are restricted to the Kmlallotype, and a disproportionate usage of V~IIIb and V~:IIa amonghumanRFswas observed (24, 56). Schlomchik et al (28) proposed,based on structural homology someVI( gene families and the exposure of these shared sequences on

Annual Reviews 336

BONA

Table 3 Representation of VKfamilies amongautoantibodies with various specificities

Annu. Rev. Immunol. 1988.6:327-358. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Specificities Thyroglobulin DNA Sm Myelin basic protein Intrinsic factor Skin antigens Thymocytes GBM RBC Total frequency

VK1 I/4 2/9 2/6

V~4

Not VK8 V~10 V~19 V~21 VK24 identified

1/4 1/6

2/4 2/9 1/6

1/3

1/6

5/9 1/6

1/3

1/3

1/1 1/1 2/6 4/10 12/43

3/6 1/10 2/43

2/10 3/43

6/43

1/6 1/1 1/10 7/43

2/i0 0/43

0/43

10/43

the surface of RF molecules, that some VI~ framework segments could contribute to the binding of Fc fragment of IgG. Crystallographic studies of the self-antigen-autoantibody complex will be required to test this hypothesis which implies that someself-antigen-autoantibody interactions are not "binding-site" mediated.

Variable Region Gene Complexes in AutoimmuneStrains As was mentioned above, the germline genes comprising the V loci are

classified in familiesbasedon DNA and proteinsequencehomology. These familiescomprisethe majorityof known polymorphic Vgermlinegenes. Thepolymorphism of theseVgenefamiliescanbe studiedusingrestriction fragmentlength polymorphism (RFLP)analysis. This methodcan reveal deletions, gene duplications, crossover, and even point mutations in the V genes and, therefore, can detect differences in the V gene germline repertoire between autoimmunestrains and their ancestors and normal strains of the same IgH V haplotype. Kofler et al (57) using RFLPanalysis

did not find importantdifferencesbetweenautoimmune strains andtheir normalancestorswith the exceptionof an additionalfragmentin the Q52 family in NZB and NZWcompared to AKRmice bearing the same haplotype (vdn). Based on this analysis, these authors concluded that the IgV gene complex in lupus-prone miceis essentiallynormalandthat the subtledifferencesobserved can be due to the rate of divergence between various strains studied. RFLPanalysis of V loci has been extended in our laboratory to other

Annual Reviews V GENES OF AUTOANTIBOD1ES

337

Annu. Rev. Immunol. 1988.6:327-358. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

autoimmune strains. HindIII and EcoRI-digested DNAwere probed with 8 VHand 8 V~ probes prototypic for each family. Several mousestrains have been used in this study. These are classified according to the VH haplotypes. V~ SJL--genetically predisposed to develop experimental demyelinizing encephalitis. BXSB--proneto develop a lupus-like syndrome. Tight skin--prone to develop a scleroderma-like syndrome. These mice live only as heterozygotes (TK+/TK-). Motheaten (meV)--prone to develop a multi-autoimmune syndrome. C57BL/6 and C57BL/6 Smn (separated from C57BL/6 5 years ago)used as normal strains not prone to autoimmunediseases. V~ 129/Sv--prone to develop RFs during aging. Balb/c--normal control strain. V~ NZBand NZW--prone to develop a lupus-like syndrome. AKR--a non-autoimmune strain. V~HMRL/lpr--a strain prone to develop a lupus and arthritis-like syndrome. C3H/He and LG--non-autoimmune strains. The data illustrated in Figures 1 and 2 and summarizedin Table 4 show that a single difference in EcoRI-digested DNAprobed with VHQPC52 was observed between the V~ strain 129/Sv and BALB/cin spite of the fact that these two strains were generated several decades ago on different continents. Two differences have been observed between NZBand NZW versus AKRin the VH QPC52 and VH S107 families (53). However, multiple differences have been observed in the VI~ families frequently used in autoantibodies. For a long time it was thought that the genes located in the Vr: locus represent a non-polymorphic genetic unit. A dimorphism of VKwas noted amongvarious mousestrains determined by an additional cysteine peptide (Ib) in tryptic mapsand by isoelectrofocusing (Igk.Ef2). The genes responsible for this polymorphismsegregate together with Ly-3 thymocytic antigen or Hd markers located on chromosome6. Southern analysis of Bam H1 RFLPbands using Vr:19 and VI~21 probes allowed definition of three haplotypes in the VI~21and two in the VI~19families (117). Studies of RFLPpatterns of eight VI~ families carried out in our laboratory by using Hind III and EcoRI digested DNAfrom various murine strains showedlack of polymorphismin Vr~22and VI~24families and two or three polymorphic haplotypes in VK1, VK4,V~8, VK10,VK19and ¥~21. It is notable that the polymorphism of VKgene families which are frequently used by autoantibodies show important differences between

Annual Reviews

Annu. Rev. Immunol. 1988.6:327-358. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

338

BONA

Figure 1 Southernblots of HindlII-digested liver DNA from eight strains SJL, C57BL/6J, motheaten (ME), NZW,AKR,NZB,BALB/cand 129/Sv mice. Each blot was hybridized with indicated Vnprobes.

normal and autoimmune strains of the same haplotype. In EcoRI and Hind III DNAdigests, additional RFLPbands are present in the TSK mutant strain, compared to C57BL/6J, when hybridized with VI~I and VK10probes. TSKshows an additional restriction fragment of 7.6 kb and probably a second band of 4 kb with a VK1probe, and an additional band of 4.4 kb is found with a V~10probe in a Hind I1~I digest. A further band of 3.7 kb is found in a Hind III digest probed with VI~19. NZBcompared to C57BL/6J, BALB/c, and NZWstrains also shows several additional bands of 7.8, 7.0, 3.6, and 1.8 kb and 0.6 kb in Hind III digests, and two

Annu. Rev. Immunol. 1988.6:327-358. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Annual Reviews V GENES OF AUTOANTIBODIES

339

~z

Annual Reviews

Annu. Rev. Immunol. 1988.6:327-358. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

340

BONA

altered DNAfragments of 4.7 kb and 5.9 kb in EcoRI digests probed with VK1. MRLcompared with AKR(same haplotype) or with LG and C3H (ancestors) also shows an additional band of 3.9 kb in Hind III digests probed with VK19. While MRLpresents a restriction pattern similar to LG and C3H in Hind III digests probed with VK1, AKRlacks bands of 5.5 kb, 4.4 kb, and 4.0 kb. SJL also shows an additional restriction fragment of 4.4 in EcoRI digests probed with VI~19 when compared to the LG mouse strain of the samehaplotype. In contrast, no differences in the restriction pattern could be seen between 129/Sv and BALB/cstrains. If the subtle differences of the VHgene can be explained by genetic divergence between strains of the same nominal haplotype (57), extensive polymorphismof VKcould indicate important alterations in the germline gene repertoire of autoimmunestrains. It is possible that "unique" germline genes present in autoimmune strains can be the source of genes rearranged in autoantibodies. Comparison of nucleotide sequences of rearranged V genes in autoantibodies with germline genes isolated from bands "unique" to autoimmunestrains and "shared" by autoimmuneand normal strains will permit us to determine whether or not there are subsets of germline genes from which V genes of autoantibodies are generated. High Idiotypic

Cross-reactivity

of Autoantibodies

Study of the idiotypy of autoantibodies has provided important information on the function and relatedness of V genes encoding self-specificities, since the idiotypes are phenotypic markers of V genes and also targets for regulatory processes (see data reviewed in 58). An important feature which emerged from the idiotype of autoantibodies that are directed against multivalent antigens is that they often share an IdX. Thus, in the human, IdXs have been identified on cold hemagglutinins, autoantibodies causing hemolytic anemia (59), rheumatoid factors (60), and antibodies specific for ss or dsDNA(61, 62). idiotype located on the kappa chain of anti-DNA antibodies in SLE patients was also identified on 87/706 myelomaproteins tested. Of these Id + myelomaproteins, 29 bound to dsDNA(63). Similarly, in murine autoimmune models, IdXs have been identified on anti-Sm (66), NZB Coombsanti-RBC (67), and rheumatoid factors (35). Furthermore, autoantibodies express interspecies IdX, indicating that the DNAsegments encoding these idiotypic specificities were conserved during phylogeny. Thus, interspecies IdX (humans, mice, and rats) were described in the case of anti-thyroglobulin antibodies (68) and anti-acetylcholine receptor antibodies (69).

Annual Reviews

Annu. Rev. Immunol. 1988.6:327-358. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

V GENES OF AUTOANTIBODIES

341

Theseobservationsstrongly suggestthat in spite of the heterogeneityof someautoantibodyresponses, the specificity of the binding sites and the idiotopes of autoantibodiesare germline-geneencoded. Studies carried out in our laboratory on a large panel of autoantibodies showedthat actually a high idiotypic cross-reactivity exists among autoantibodies with various specificities (70). These studies havebeen carried out using four IdX systems: LPS10-1IdXexpressed by an RF, 62Id on anti-thyroglobulin monoclonalantibodies obtained from BALB/c mice, Y-2 IdX expressed on an anti-Sm, and H130IdX on an anti-DNA antibody obtained from MRL/lprmice. Thedata presented in Table 4 showthat the IdXof these antibodies can be sharedwith autoantibodieswith various specificities. Themoststriking feature of this study was the independenceof the expression of these idiotypes and Vnor Vr: usage in autoantibodies(35, 53). Since weagree that the ingredients of autoimmunity,self-antigens and autoreactiveclones are present in eachindividual, suppressorT cells could play an importantrole in the maintenanceof self-tolerance undernormal conditions. Indeed, neonatal thymectomy or repeated low doses of cyclophosphamide increased the susceptibility of animalsto spontaneousdevelopmentof thyroiditis or encephalomyelitis(71, 72). Furthermore,Moore & Calkins (73) showedthat T cells that maycontrol the anti-mouseRBC response in young NZBmice are lost in old Coombspositive mice. Two types of suppressor T cells can be involved in this process: antigen or idiotypespecific T cells. If, for somecirculatingautoantigens,it is easyto explain howantigen-specific T cell clones are continuouslyexpanded,it is difficult to see howsuppressorT cells specific for sequesteredantigensare stimulated. Therefore,it appearsthat in the case of non-organspecific or sequesteredautoantigen-specificclones, idiotype-specificsuppressorT cells mayplay an important role in the maintenanceof tolerance. IdX shared by clones producingautoantibodieswith various specificities (particularly against sequestered antigens such as DNA or Sm)can be the target of naturally occurring idiotype-specific suppressor T cells. Wepreviously describedthe waythat naturally occurringidiotype-specific suppressorT cells are responsiblefor the control of expressionof someminoror silent idiotopes (74, 75). The elimination of such suppressor cells wasaccompaniedby an increased expressionof minoror silent clones (76). If the immune systemis sustained at a steady state by an equipoise of lymphocyteclones bearing complementaryreceptors, one mayenvision that the equilibrium betweenautoreactive clones and idiotype-specific suppressor T cells can be upset by a variety of mechanisms:surface expressionof the sequesteredantigens mediatedby buddingviruses, expansion of autoreactive clones by stimulation with cross-reactive antigens or

Annu. Rev. Immunol. 1988.6:327-358. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Annual Reviews 342 ~o~a

Annual Reviews

Annu. Rev. Immunol. 1988.6:327-358. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

V GENES OF AUTOANTIBODIES

343

by anti-idiotypic antibodies. Minuteamountsof anti-idiotypic antibodies can stimulate the expansionof clones bearing the sameidiotype (77, 78). Antibodiesfrom myasthenicmothers transferred via the placenta to the newborncan cause not only the muscle weakness symptomatologybut also the stimulation of anti-Id antibody production whichexpandsthe clones producinganti-acetylcholinereceptor antibodies (79). Monestieret al (43) studied the expression of idiotypes specific for foreign antigens on autoantibodieswith various specificities (DNA,thyroglobulin, myelinbasic protein, rheumatoidfactor). This study wascarried out on a panel of 20 monoclonalautoantibodies encoded by genes from the Via J558 family, since wehave available in our laboratory several idiotypic systemsof Vr~J558antibodiesspecific for foreign antigens. In this study, the followingidiotypic systemshave beeninvestigated: J558 IdX of anti-al ~ 3 dextran antibodies, Py206and Py211IdX of antibodies specific for influenza virus hemagglutinin,cGAT,a cross-reactive idiotype expressed on anti-GATpoly (Glu, Ala, Tyr) and the CRI anti-phenylarsonateantibodies. Thesummary of the data obtained from the competitive inhibition RIA usinglabeled idiotypes andmonoclonal anti-Id antibodiesis illustrated in Table5. Thesedata clearly showthat, taking into accountour small panel of monoclonalautoantibodies, an important fraction of themshare the IdXof VHJ558÷ antibodiesspecific for foreign antigens. Since anti-idiotype antibodies can stimulate clones with various specificities that bear regulatoryidiotopes(80) througha cross-regulatingidioo type-mediatedmechanism,one mayimaginethat autoanti-idiotypic antibodies produced during conventional immuneresponses (subsequent to infectious diseases)could stimulate autoreactiveclonesfavoringthe breaking of self-tolerance. Thus,anti-idiotypic antibodiesspecific for regulatory idiotopes could expandsilent or minor clones to becomedominant.This wasclearly demonstratedin the case of Id62, a markerof silent or minor anti-thyroglobulin clones in BALB/cmice. The administration of antiId62 antibodiessignificantly stimulatedthe proliferation of clonessharing Id62 regulatoryidiotypes (81). Similarly,rabbit antibodieselicited by the immunizationwith a monoclonalanti-Id antibody specific for a multiorgan-specificantibody(82). Othertypes of anti-idiotypic antibodiescan also contributeto the breaking of self-tolerance. Abundantdata demonstratethat anti-Id antibodies carry the internal imageof self-antigenssuchas insulin (83, 84), TSH(85), RF(86), and hormonereceptors (87). Suchanti-idiotypic antibodies be responsible for the pathogenicevents contributing to type I diabetes (84), Grave’sdisease (85), or asthmaticsyndromes (88). Anti-idiotypic antibodies carrying the internal imageof self-antigens

Annu. Rev. Immunol. 1988.6:327-358. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Annual Reviews 344 aO~A

Annual Reviews

Annu. Rev. Immunol. 1988.6:327-358. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

V GENES OF AUTOANTIBODIES

345

can stimulate autoreactive clones in lieu of autoantigens. Similarly, epibodies, anti-Id antibodies that also exhibit binding properties to selfantigens (89), can play a role in the pathogenesisof autoimmune diseases. In this respect, it should be mentionedthat a large fraction of RFsfrom patients with cryoglobulinemia exhibit epibodyproperties since they bind to Fab fragments of IgG isolated from immunecomplexesas well as to Fc fragments(90, 91). Holmdahlet al (92) recently obtained a monoclonalantibody with epibody properties from mice immunizedwith collagen type II-antibody complex.This epibody binds to Fab fragments of anti-collagen type II antibody and to Fc fragments. This type of epibody mayrepresent a link between anti-collagen and RFproducing clones and could play an important role in immunologicphenomena in rheumatoidarthritis. Theseobservationssuggestthat various types of autoanti-Id antibodies of Ab2a,r, or e types can contribute to the breakingof self-tolerance. If under certain conditionssuch antibodies can be responsible for the onset or perpetuationof autoimmunity,probablyin the majority of the diseases, they do not suffice to initiate the disease whichinvolvescompleximmunological mechanisms.Indeed, other factors are important in autoimmune diseases. Thus,autoantigens,particularly those that are thymusdependent, should be recognized by helper T lymphocytesand should require the expressionof class Ii antigens on the surface of somaticcells. Thereare data that demonstratethe derepressionof genescodingfor class II on the cells that are targets of autoimmune diseases(data reviewedin 93). Genetic factors also havean importantrole in the occurrenceof autoantibodiesin both humanand animal autoimmunedisease. Myastheniagravis is one of the best examplessince it is associated with the B8and DR3haplotypes in caucasoids. The production of autoanti-RNAantibodies (RO/SSA and La/SSB)appears to be associated with DQ1and DQ2alleles in primary Sjogren’s syndrome, and the gene complementationof transassociated productscontrols high concentrationsof these autoantibodies(94). Multispecific

Binding Properties of Autoantibodies

Theother side of molecularstudies is investigations of immunochemical characteristics of the combiningsite of autoantibodies. There are three major mechanismsby whichautoantibodies can contribute to the pathogenesis of autoimmune diseases (93): (a) by interacting with target cells, they cause damagingeffects which finally lead to the replacementof destroyedcells by connectivetissue; (b) by interacting with the cell receptor, they can block the binding

Annual Reviews

346

BONA

Annu. Rev. Immunol. 1988.6:327-358. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

biologically active substancesimportantfor the function of a particular organ; (c) by interacting with the receptor of certain cells, they can stimulate the target cells in lieu of hormones andalter cell functions. Table 6 depicts humanautoimmune diseases in whichthese three types of mechanismsare involved in pathogenesis. Oneof the mainunsolvedquestions is the nature of the antigen which contributes to the expansionof clones producingautoantibodies. Whether self-reactive clones are triggered by internal or environmental antigens is still an open question. Ananswerto this question perhaps can be found in the studies on the bindingproperties of autoantibodies. Thedevelopmentof the hybridomatechnologyallowed accurate studies on the binding properties of autoantibodies. Thesestudies led to a surprising observationindicating that there are antibodiesspecific for foreign antigens that can bind to self-antigens, autoantibodiesthat bind to foreign antigens, and antibodies that bind not only to ubiquitous autoantigens, but also to autoantigens knownto be involved in autoimmune diseases. This multiple binding property does not represent a simple experimental error related to the use of highly sensitive techniques(RIA,ELISA)with antigens immobilized on solid surfaces. Competitive inhibition and measurements of the affinity of these antibodies clearly indicate that this is a property of the combiningsite of antibodies that could be important in autoimmunephenomena. Antibodies Specific for Foreign Antigens That Bind to Autoantigens After it wasobservedthat patients with acute rheumaticfever haveantibodiesreacting with heart tissue (95), these antibodieswerealso shown react with brain (96) and skeletal muscle(97). Becauseof the polyclonal nature of antibodies, immunochemical studies aimedat defining the fine Table 6 Effects of organ specific autoantibodies in various humanautoimmune diseases Destructive Hashimoto’sthyroiditis Fundalandantral gastritis Pemphigusvulgaris Vitiligo Autoimmune diabetes Gonadalfailure Autoimmune hemolytic anemia

Blocking

Stimulatory

Myastheniagravis Insulinresistant diabetes Addison’sdisease Asthma(somecases)

Grave’sdisease AdrenalCusb_ing’s disease

Annual Reviews

Annu. Rev. Immunol. 1988.6:327-358. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

V GENES OF AUTOANTIBODIES

347

specificity of such antibodies have been difficult. However,these observations have been confirmed by studies using monoclonal antibodies. Thus, it was shown that MAbsobtained from BALB/cmice immunized with Streptococcus pyogenes M, type 5, also bind to myosin, actin, or actinin (98). Kabat et al (99) showedthat a myelomamacroglobulinspecific for (2-8) poly-N-acetyl neuraminic acid of the capsular polysaccharide group B meningococci and of E. coli K1 binds to denaturated DNAor polynucleotides. In another study, Naparstek et al (100) found that monoclonal macroglobulins specific for Klebsiella polysaccharides bind to DNAand that some of them share a cross-reactive idiotype (16/6) expressed on lupus anti-DNA antibodies. A comparison was made of the primary structure of the first 40 N-terminal amino acids of the light chain of one macroglobulin that expresses the 16/6Id and binds to Klebsiella, and to DNA with an anti-DNA 16/6Id monoclonal antibody. The comparison showed only a single aminoacid residue difference in CDRI,indicating that similar VLgenes are used by anti-Klebsiella and anti-DNAantibodies. Recently, we have studied the binding properties of a panel of 25 hen egg lysozyme (HEL) monoclonal antibodies to a variety of autoantigens knownto be involved in autoimmunediseases. Three HEL-specific antibodies bound to DNAand one to intrinsic factor as assessed by direct binding and competitive inhibition RIA. Furthermore, the measurements of equilibrium constants showedthat the affinities of these antibodies for dsDNAand lysozyme were quite similar. The antibody response against HELis quite heterogeneous, and clones secreting antibodies use genes from various Vr~ families. Interestingly, all anti-HEL antibodies exhibiting self-binding properties are encoded by genes from the Vr~ 36-09 family and share a cross-reactive idiotype. Sequences of the V~ genes of these antibodies exhibit a high degree of homology. Our results are similar to those reported by Naparstek et al (101) who showedthat a small fraction of anti-arsonate antibodies encoded by genes from the Vr~ 36-65 family and expressing CRI bind to ssDNA. These experimental results indicate that autoantibodies (a) to platelets found in patients with rubella (102), (b) to heart in patients with Streptococcus infections (94, 95, 96), (c) to DNAin patients with syphilis leprosy (103), or rheumatoid factors in patients with acute or chronic infectious diseases (data reviewed in 104) could be related to activation autoreactive clones by environmental antigens. A similar explanation can be advanced for the appearance of autoantibodies following a variety of infections such as vaccinia, reovirus, inflenza, Sendai, or retroviruses (data reviewed in 105).

Annual Reviews

348

BONA

This conceptis in agreementwith data showingthat an intestinal infectious agent is responsible for the production of rheumatoidfactors in 129/Sv mice. The synthesis of RFsin old 129/Sv mice was completely preventedby caesarean-derivationandrearing the micein isolators (106).

Annu. Rev. Immunol. 1988.6:327-358. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Autoantibodies That Bind to ForeignAntigens Since antibodiesto foreign antigens clearly can exhibit self-reactivity, we addressed the question in a recent study of whether or not bona fide autoantibodies can bind foreign antigens (43). In this study, wechose group of 20 autoantibodies, including antibodies specific for DNA or Sm obtainedfromMRL/Iprmice, antibodies specific for collagen type II from immunizedDBA/1mice, antibodies to thyroglobulin from immunized CBA mice, and antibodies specific for myelinbasic proteins from SJLand v me mice. All of these autoantibodies are encodedby genes from the VH J558 family. Westudied the binding of these autoantibodies to foreign antigens knownto be recognized by VHJ558+ antibodies. These antigens includeddextrans, lipopolysaccharides,synthetic polypeptides[poly (Glu, Ala, Tyr), poly (Glu, Tyr), poly (Glu, Lys), po!y (Glu, Phe)], influenza viruses, lysozyme,arsonate, and NP. Of 20 autoantibodies 9 boundto at least 1 of the foreign antigens, particularly to synthetic antigens such as poly (Glu, Tyr) and poly (Glu, Phe). Twoanti-DNAantibodies obtained from MRL/lprmice showedsignificant binding to other antigens such as lysozyme,arsonate, and E. coli or S. providenciaelipopolysaccharides. The Kd of monoclonalautoantibodies for autoantigens and for foreign antigens were quite similar, ranging between10-4 and 10-7. Noimportant differences in Kd for foreign and autoantigens were observed between autoantibodies obtained from animals with autoimmunediseases and those immunizedwith autoantigens in FCA(Table 7). Datademonstratethat autoreactive B cells can be stimulatedin vitro to produce autoantibodies. Thus, spleen cells from MRL/lprincubated with purified Smantigens developa significant PFCresponse after 4 days in culture (106). Lymphocytesfrom patients with autoimmunethyroiditis can be stimulated to produceIgGanti-TGantibodies using insolubilized TG(108). However,the findings discussed above regarding the ability of autoantibodies to bind to foreign antigens, and of antibodies elicited by immunizationwith foreign antigens to bind to self-antigens, strongly suggest that autoreactiveclones can be activated by internal (autoantigens)as well as by environmental(foreign) antigens. Several mechanismscan be envisioned. The simplest maybe related to a trivial cross-reactivity betweenforeign andself antigens. Thereare data indicating sequencehomology betweenproteins of viruses causing neuritis

Annual Reviews V GENES OF AUTOANTIBODIES Table 7 Binding properties Monoelonal antibodies 10 #g/ml

349

+ b"bona fide" autoantibodies to foreign antigens of VHJ558

Bindingto plates coated with

Percentageof inhibition of binding 15 ngantigen/well

Kd(g/ml)

MBP Glib

Annu. Rev. Immunol. 1988.6:327-358. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

15-32

UN59-9

MBP GLq5 MBP GT GLq~ Lys

a14,439+606 955_+158 10,611+1328 5570+1008 5605+307 2873+720

-5 5.2 x 10 0

63% 0 MBP

61.7% 72% GLib

53.5% 77% 62.5% 55% Sm

83.6%

GLib

GT 50.9% 82.6%

GT

Lys

86.5% 88.1%

54.6%

-4 1.2 x 10 9.4 x 10 5 -5 1.0 x 10 -5 8.9 × 10

70.8% 60.6%

-5 6.6 x 10 -5 7.6 x 10 -5 8.0 × 10

Sm GT GLq~

1925+196 1927+134 2597_+106

55.3% 73.3% 80.7% Sm

58.2%

Y-12

Sm GAT

2145+482 1710__+307

42.4% 79% TG

40.6% 76.6% GT

1.8 x 10 4 -5 6.5 x 10

81BI

TG GT

6537_+696 5070_+347

76,3% 74.4% TG

84.8% 88.2% GL~

-5 7,8 x 10 -5 7.8 x l0

62.3% 52.5% 66.7% G2a

80.3% 91.4% GLib

GT

54.3% 59.6% 65.5% DNA

53.1% 66% 68.6% GLib

46.5% 70.1% 71.2% GT

50.9% 70% 20% 86.3% 58.3% 84% DNA

78.4% 79.7% 52.9%

83.8% 73.6%

GT

E. coli

S. prov.

75.8% 86% 61% 58.3% 58.7%

77.2% 44% 81.3%

65% 91.7%

66.5% 74.5%

6B6

84A3

LPSS-4

HI30

H102

TG GT GLib G2a GT GLib DNA Card GLib GT Lys Ars DNA Card GT E. coli S.prov.

2203_+640 2905_+654 4908_+307 1083±134 1762_+62 1811±117 1672_+62 11,231+_ 3932 16,431-+572 8089_+511 6643_+486 6742_+1472 4405+__70 10,888-+4848 3451_+407 1215_+3 1167_+173

86.1% GAT

GT -4 1.2 × 10 -5 7.0 x 10 -5 7.8 x 10

80.6% 81.9%

-4 1.2 x 10 -5 4.5 x 10 -5 8.0 x 10 Lys

Ars

60% 90.6%

67.6% 94.2%

83% 55.3% 83.5%

53.7% 70.8%

cpm--averageof triplicate + SD. Abbreviations:Lys= Lysozyme, Ars = Arsonate,S. prov. = S. providencia,Card~ eardiolipin. ND= not done.

1.2 -4 × 10 cND -5 6.6 x 10 -4 1.2 x 10 -4 1.6 x 10 -4 5.3 x 10 -6 1.4 × 10 ND -5 5.3 x 10 -5 5.2 x 10 1.6 × 10 6

Annual Reviews

Annu. Rev. Immunol. 1988.6:327-358. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

350

BONA

and myelin proteins (109). The other possibility to be considered is molecular mimicryin whichfunctional groups interacting with the lymphocytic receptor can be shared by completely unrelated foreign and autoantigens. Finally, it is also possiblethat idiotypic regulatoryprocessescan play a role. If the receptorof clonesproducingantibodiesspecific for foreignand autoantigensshare a cross-reactiveidiotype, idiotype-specifichelper T cells for anti-idiotype-producing B cells expandedsubsequentto immunization with foreign antigen (e.g. an infectious agent) can activate autoreactive clones. Further experimentsare required both to provide direct evidence that a clone producingantibodies able to bind to self andforeign antigens can be activated by a foreign antigen or anti-idiotypic antibodies and to producepathogenic autoantibodies. Autoantibodies Bindin# to Multiple Self-Anti#ens Numerous observationsduring the past five years indicate the existence in the B cell repertoire of clonesable to produceantibodiesexhibitingmultiple bindingspecificities (42, 103). Dighieroet al (109) foundthat a high proportion (6.25%)of hybridomas obtained fromnewbornBALB/c mice exhibit multiple binding specificities for self-antigens. Holmberg et al (111) have shownthat there is a high idiotypic connectivity amongsuch antibodies. Notkin’s group also demonstratedthe existenceof antibodiesthat can bind to varioustissue-specific antigens that have been designated as multi-organ-specific antibodies (112). In our studies wecharacterized multispecific antoantibodies produced by hybridomasobtained from LPS-stimulated spleen cells from BALB/c and NZBmice, from 1 and 2 month old motheaten mice, from nu/nu C57BL/6,and from 1 month old MRL/Iprmice injected with anti-Id antibodies. Theseantibodies can bind to two or several autoantigens such as Fc, intrinsic factor, thyroglobulin, myelinbasic protein, DNA, collagen type II, AchR(Table 8). Frommolecular and immunochemicalstudies of our panel of multispecific autoantibodies, the following information emerged: (a) the vast majorityofmultispecificautoantibodiesare of the IgMisotype; (b) the high frequencyofmultispecific autoantibodies amonghybridomas obtained from motheatenmice suggests that the precursor of clones producingsuch antibodies could belong to the LyI.Bsubset; (c) the presenceof clonesproducingsuch antibodiesis an intrinsic property of the B cell repertoire whichis not underT cell control. Hybridomas producing multispecific antibodies have been obtained from nu/nu C57BL/6mice (107);

Annu. Rev. Immunol. 1988.6:327-358. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Annual Reviews V GENES OF AUTOANTIBODIES

"~b 0

351

Annual Reviews

Annu. Rev. Immunol. 1988.6:327-358. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

352

BONA

(d) precursorsof multispecific antibodyformingcells are found in murine strains prone to autoimmunediseases (motheaten, NZB,MRL/lpr), well as in normalstrains; (e) the bindingof someantibodies to various antigens can be inhibited a single or only a few antigens, whereasthe binding of others can be reciprocally inhibited by all the antigens to whichthey bind (53, 113); (f) the multispecific antibodies use genes fromall Vr~families, but the usage of 3’ families is higher (114) comparedto an LPS-stimulated population(115); (g) our data showeda bias in the usageof V~families whichare generally preferentially used(VI~I, Vr:8, VI~10,Vr:19)by autoantibodies. Whateveris the immunochemical basis of multiple binding of autoantigens by someantibodies, their physiological and pathological implications are not yet known.They could have a physiological role in clearing metabolites, as was proposedby Grabar (12), or they mayprevent autoimmunity by blinding the immunesystem to environmentalepitopes cross-reactive with self epitopes(116), Alternatively,such multispecificantibodiesof the IgMisotype can acquire high affinity for a single antigen, subsequentto somatic mutations during clonal maturation, and therefore, they could becomepathogenic. Whetheror not these interpretations are correct remainsto be seen. Conclusions Strong evidence suggests that antibodies produced during someautoimmunediseases exhibit pathogenic effects and, injected into animals, induce an autoimmunesymptomatology. Studies of the characteristics of V genes encodingautoreactive antibodies might provide clues for the further understandingof autoimmune diseases. Theorganization of genes encodingautoantibodies does not appear to differ in anygeneral wayfromthose specific for foreign antigens. Restriction enzymaticlength polymorphism analysis ofV germlinegenes of autoimmune strains, comparedto normalstrains of the samehaplotype, showonly subtle differences in VHgene families betweensomeautoimmune and normal strains. A moreevident polymorphismwas observed in some V~families(VI~1, VI~10, VI~I9) whichare frequentlyusedby autoantibodies with various specificities. FourV~:families (VK1,VI~4,V~10,and V~:19) are used by 80%of autoantibodies secreted by hybridomasobtained from mev mice, nearly all B cells of these miceexpress Lyl.B marker,and 60% of hybridomasproduce autoantibodies. A biased usage of 3’ and VH families wasalso observedin autoantibodieswith various specificities.

Annual Reviews

Annu. Rev. Immunol. 1988.6:327-358. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

V GENES OF AUTOANTIBODIES

353

These data suggest that autoimmune repertoire is generated by a limited number of Vr~ and VI~ gene families. Immunochemical studies of autoantibodies suggest that they exhibit multibinding specificities to self and foreign antigens. In addition they show cross-reactive idiotypes with antibodies specific for foreign or self antigens. Since there is no strong evidence that the break of self-tolerance and the expansion of autoreactive clones is driven by autoantigens, it is possible that the ability of autoantibodies to bind foreign antigens can explain the activation of autoreactive clones. High idiotypic connectivity among autoreactive clones suggests that mechanisms upsetting the idiotype network also can be involved in the pathogenesis of autoimmune diseases. ACKNOWLEDGMENT This work was supported by Grant Number 2092 from the Council Tobacco Research, USA, Inc.

for

Literature Cited 1. Burnet, F. M.1959. The Clonal Selection Theoryof Acquiredlmmunity.London: CambridgeUniv. Press 2. Hooper, B., Whittingham, S., Mathews, J. D., Mackay, I. R., Curnow, D. H. 1972. Autoimmunity in a rural community. Clin. Exp. Itnrnunol. 12:79-87 3. Roberts, I. M., Whittingham, S., Mackay,I. R. 1973. Lancet 2:936-40 A., Mon4. Bellon, B., Manheimer-Lory, estier, M., Moran,T., Dimitriu-Bona, A., Alt, F., Bona,C. 1987. Highfrequency of autoantibodies bearing cross-reactive idiotopes amonghybridomas using Vn7183genes prepared from normal and autoimmunestrains. J. Clin. Invest. 79:1044-53 5. Nossal, G. J. V., Pike, B. L. 1978. Mechanism of clonal abortion tolerogenesis. I. Responseof immaturehapten-specific B lymphocytes.J. Exp. Med. 148:1161-70 6. Nossal, G. J. V. 1983. Cellular mechanismsof immunologic tolerance. Ann. Re~. Immunol.I: 3342 7. Welch,M.J., Fong,S., Vaughan, I. H., Carson, D. A. 1983. Increased frequencyof rheumatoidfactor precursor B lymphocytesafter immunizationof normal adults with tetanus toxoid. Clin. Exp. Immunol.51:299-304

8. Manheimer-Lory,A. J., Bona, C. A. 1986. Are rheumatoidfactors produced during conventional immune responses beneficial to the host? In Paradoxesin Immunology,ed. C. W.Hoffman,J. G. Levy, G. T. Nepom,pp. 123-35. Boca Raton, Fla: CRC 9. Nemazee, D. A., Sato, V. L. 1983. Induction of rheumatoid antibody in the mouse: regulated production of autoantibody in the secondary humoral response.J. Exp. Med.158:529--45 10. Manheimer,A. J., Victor-Kobrin, C., Stein, K. E., Bona, C. A. 1984. Antiimmunoglobulinantibodies. V. Agedependentvariation of clones stimulated by polysaccharideTI-2 antigens in 129 and MRLmice spontaneously producinganti-y-globulin antibodies. d. Immunol.133:562-68 11. Dighiero, G., Guilbert, B., Avrameas, S. 1982.Naturallyoccurringantibodies against nine common antigens in humansera. II. Highincidence of monoclonal Ig exhibiting antibodyactivity against actin and tubulin and sharing antibodyspecificities withnatural antibodies. J. Immunol.128:2778-92 12. Grabar, P. 1983. Autoantibodies and physiological role ofimmunoglobulins. Immunol. Today 14:337-40 13. Khansari, N., Fudenberg,H. H. 1984.

Annual Reviews

Annu. Rev. Immunol. 1988.6:327-358. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

354

BONA

Physiologicalrole of autoantibodies. Immunol.Today 5:87 14. Schoenfeld,Y., Schwartz,R. S. 1984. Immunologicand genetic factors in autoimmune diseases. N. Engl. J. Med. 311:1019-29 15. Knight, J. G. 1982. Autoimmune diseases: defects in immunespecificity rather than a loss of suppressorcells. Immunol.Today 3:326-28 16. Bona,C., Stein, K. E., Lieberman,R., Paul, W.E. 1979. Direct and indirect suppression induced by anti-idiotype antibodyin the inulin-bacterial levan antigenic system. Mol. ImmunoL16: 1093-1101 17. Diamond,B., Scharff, M. D. 1984. Somatic mutation of the T15 heavy chain gives rise to an antibody with autoantibodyspecificity. Proc. Natl. Acad. Sci. USA81:5841-44 18. Nye,L., de Carvellio,L. P., Roitt, I. M.1981. Aninvestigation of the clonality of human autoimmunethyroglobulin antibodies and their light chains. Clin. Exp. Immunol.46:161-70 19. Brooks, M. S., Aldo-Benson,M. 1986. Defectsin antigen specific immune tolerance in continuousB cell lines from autoimmune mice. J. Clin. Invest. 78: 784-89 20. Theophilopoulos, A. N., Shawler, D. L. O., Eisenberg, R. A., Dixon,F. J. 1980. Splenic immunoglobulin secreting cells andtheir regulation in autoimmunemice. J. Exp. Med. 151: 44666 21. Montsopoulos,H. M., Boehm-Truitt, M., Kassan,S. S., Chused,T. M.1977. Demonstrationof activation of B lymphocytes in NewZealand black mice at birth by immunoradiometricassay from murine IgM. J. Imrnunol. 119: 1639-44 22. Capra, D. J., Kehoe,M., Williams,R. C., Feizi, T., Kunkel,H. G. 1972.Light chain sequence of human IgM cold agglutinins. Proc. Natl. Acad.Sci. USA 69:40-43 23. Kunkel, H. G., Winchester, R. J., Joslin, F. G., Capra,J. D. 1974.Similarities in the light chains of anti-~globulins showingcross-reactivespecificities. J. Exp. Med.149:128-36 24. Goni, F., Chert, P. P., Pons-Estel,B., Carson, D. A., Fragione, B. 1985. Sequencesimilarities and cross-idiotypic specificity of L chains among humanmonoclonalIgMwith anti-~,globulin activity. J. Immunol.135: 4073-79 D., 25. Chert, P. P., Fong,S., Normanse11, Houghten, R. A., Karras, J. G.,

Vaughan,J. H., Carson, D. A. 1984. Delineationof a cross-reactiveidiotype on humanautoantibodies with antibodyagainst a synthetic peptide. J. Exp. Med. 159:1502-11 26. Chen,P. P., Albrant, K., Radoux,V., Chen,E. W.,Schrantz,R., Liu, F., Carson, D. A. 1986.Geneticbasis for the cross-reactive idiotypes on the light chains of humanIgM anti-IgG autoantibodies. Proc. Natl. Acad.Sci. USA 83:8318-22 27. Capra,J. D., Kehoe,J. D. 1974.Structure of antibodieswithsharedidiotypy: The complete sequence of the heavy chain V-regions of two IgManti-2globulins. Proc. NatLAcad. Sci. USA 71:4032-36 28. Shlomchik, M. J., Nemazee, D. A., Sato, V. L., van Snick, J., Carson,D. A., Weigert, M. G. 1986. Variable region sequence of murine IgMantiIgG~monoclonalautoantibodies (rheumatoid factors): a structural explanation for the high frequency of IgManti-IgG B cells. Z Exp. Med.164: 407-27 29. Schlomchik, M., Nemazee, D., van Snick, J., Weigert, M.1987. Variable region sequences of murine IgMand IgG monoclonalautoantibodies (rheumatoid factors). II. Comparisonof hybridomas derived by lipopolysaccharide stimulation and secondary protein immunization. J. Exp. Med. 165:970-88 30. Reininger,L., Oilier, P., Poncet,P., Kaushik,A., Jatton, J. C. 1987. Novel V genesencodevirtually identical variable regions of six murinemonoclonal anti-bromelain-treated red blood cell autoantibodies.J. Imrnunol.138: 31623 31. Andrews,D. W., Capra, J. D. 1981. Completeaminoacid sequenceof variable domains from two monoclonal humananti-gammaglobulins of the Wacross-idiotypic group: suggestion that the J segmentsare involvedin the structural correlate of the idiotype. Proc. Natl. Acad. Sci. USA78: 37993803 32. Newrick,M. N., Ostberg, L., Wasserman,R. W., Capra, J. D. 1987. Human rheumatoidfactors of the Waidiotypic family appear to use highly homologous variable regions as humananticytomegalovirusantibody. Fed. Proc. 46:916 33. Kofler, R., Noonan,D. J., Strohal, R., Balders,R. S., Moller,N. P. H., Dixon, F. J., Theo!3hilopoulos,A, N. 1987. Molecularanalysis of the murinelupus-

Annual Reviews

Annu. Rev. Immunol. 1988.6:327-358. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

V GENES OF AUTOANTIBODIES associatedanti-self response:involvement of a large numberof heavy and light chain variable regiongenes. Eur. J. Immunol.17:91-95 34. Kofler, R., Noonan,D. J., Levy,D. E., Wilson,M.C., Moiler,N. P. H., Dixon, F. J., Theophilopoulos, A. N. 1985. Genetic elements used for a murine lupus anti-DNA autoantibody are closely related to those for antibodies to exogenousantigens, d. Exp. Med. 151:850-51 35. Manheimer-Lory,A. J., Monestier, M., Bellon, B., Alt, F. W., Bona, C. A. 1986.Fine specificity, idiotypyand nature of cloned Va genes of murine monoclonal rheumatoid factor antibodies. Proc. Natl. Acad.Sci. USA83: 8293-97 36. Kabat,E., Wu,T. T., Reid-Miller,M., Perry, H. M., Gottesman,K. S., ed. 1987. Sequence of Immunoglobulin Chains. Washington, DC: US Dept. Health Hum.Serv. 37. Brodeur, P. H., Riblet, R. 1984. The immunoglobulinheavy chain variable region (Igh-V) locus in mouse.I. One hundred Igh-V genes comprise seven families of homologous genes. Eur. J. Imrnunol. 14:922-30 38. Edelman,G. M., Olins, D. E., Gaily, J. A., Zinder, N. D. 1963. Reconstitution of immunologic activity of interaction of polypeptide chains of antibodies. Proc. Natl. Acad.Sci. USA50:753-56 39. Arant, S. E., Griffin, J. A., Koopman, W. J. 1986. Vn gene expression is restricted in anti-IgG antibodies from MRLautoimmunemice. J. Exp. Med. 164:1284-1300 40. Clarke, S. H., Huppi,K., Ruezinsky, L., Staudt, L., Gerhard,W., Weigert, M.1985.Inter andintraclonal diversity in the response to influenza hemagglutinin. J. Exp. Med.161:687-704 41. Moran,T. M., Monestier, M., Lai, A. C. K., Norton, G., Reale, M. A., Thompson,M. A., Schulman, J. L., Riblet, R., Bona, C. A. 1987. Characterization of variable region genes and shared cross-reactive idiotypes of antibodiesspecific for antigensof various influenza viruses. Viral Immunol. 1:1-12 42. Agnello,V., Arbetter, A., Ibanez, A., de Kasep,G., Powell, R., Tau, E. M., Joslin, F. 1980.Evidencefor a subset of rheumatoidfactors that crossreact with DNA-histoneand have a distinct cross-idiotype. J. Exp. Med.151: 151427 43. Monestier,M., Bonin, B., Migliorini, P., Dang,H., Datta, S., Kuppers,R.,

355

Rose, N., Maurer,P., Talal, N., Bona, C. 1987. Autoantibodies of various specificities encodedby genesfromthe V~J558familybind to foreign antigens andshare idiotopesof antibodiesspecific for self and foreign antigens. J. Exp. Med. 166:1109-24 44. Bona, C. A., Finley, S., Waters, S., Kunkel, H. G. 1982. Anti-immunoglobulinantibodies. III. Properties of sequentialanti-idiotypic antibodies to heterologousanti-2-globulins. Detection of reactivity of antiidiotype antibodies with epitopes of Fc fragments (homobodies)and with epitopes and idiotopes(epibodies). J. Exp. Med. 156:986-99 45. Bona, C. A., Goldberg, B., Metzger, D., Urbain, J., Kunkel, H. G. 1984. Anti-immunoglobulin antibodies. IV. Crossreaction of antiidiotypic antibodies specific for rabbit and murine anti-Ia~ allotype antibodies with Fc fragment of humanimmunoglobulins. Eur. J. Immunol.14:548-52 46. Trepicchio, W., Barrett, K. J. 1987. Eleven MRL/lpranti-DNAautoantibodies are encodedby genes from four VHgene families: a potentially biased usage of V~genes. J. Imrnunol.138: 2323-31 47. Yancopoulos,G. D., Desiderio, D. V., Paskind,M., Kearney,J. F., Baltimore, D., Alt, F. W.1984.Preferential utilization of the mostJHproximalVngene segmentsin pre-Bcell lines. Nature311: 757-83 48. Perlmutter, R. M., Kearney, J. F., Chang,S. P., Hood,L. E. 1985. Developmentally controlled expression of immunoglobulin VH genes. Science 127: 1597-1600 49. Herzenberg,L. A., Stall, A. M., Labor, P. A., Sidman, C., Moore, W. A., Parks, D. R., Herzenberg,L. A. 1986. TheLyl.Bcell lineage. Immunol.Rev. 93:81-102 50. Hayakawa,K., Hardy, R. R., Parks, D. R., Herzenberg, L. A. 1983. The "Ly. 1 B" cell subpopulationin normal, immunodefective and autoimmune mice. J. Exp. Med.157:202-18 51. Sidman,C. L., Shultz, L. D., Hardy, R. R., Hayakawa,K., Herzenberg, L. A. 1986. Production of ÷immunoglobulin isotypes by Ly.1 B ceils in viable motheatenand normal mice. Science 232:1423-25 52. Hardy, R. R., Hayakawa, K. 1986. Developmentand physiology of Lyl.B and its humanhomologue Leu-l.B. lmmunol.Rev. 93:53-79 53. Painter, C., Monestier,M., Bonin,B.,

Annual Reviews

Annu. Rev. Immunol. 1988.6:327-358. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

356 BOr~A Bona, C. A. 1986. Functional and molecularstudies of Vgenes expressed in autoantibodies. Immunol.Rev. 94: 75-98 54. Sidman, C. L., Marshall, J. D., Masiello, N. C., Roths, J. B., Shultz, L. D. 1984. NovelB cell maturation factor from spontaneouslyautoimmune viable motheaten mice. Proc. Natl. Acad. Sci. USA81:7199-7202 55. Kleinfeld, R., Hardy,R. R., Tarlinton, D., Dang, J., Herzenberg, L. A.; Weigert, M. 1986. Recombination betweenan expressed immunoglobulin heavychain gene and a germlinevariable gene segmentin a Ly.1 + B cell lymphoma.Nature 132:843-46 56. Adams, D. D. 1983. Autoimmune Mechanismsin AutoimmuneEndocrine Disease, ed. T. F. Davies, pp. 1-39. NewYork: Wiley 57. Kofler, R., Perlmutter, R. M., Noonan, D. J., Dixon,F. J., Theophilopoulos, A. N. 1985. Ig heavy chain variable region genecomplexof lupus miceexhibits normalrestriction fragmentlength polymorphism. J. Exp. Med.162: 346-51 58. Bona, C. 1987. RegulatoryIdiotypes. NewYork: Wiley 59. Williams,R. C., Kunkel,H. G., Capra, J. D. 1968. Antigenic specificities related to cold agglutininactivity of M globulins. Science 161:379-81 60. Kunkel, H. G., Agnello, V., Joslin, F. G., Winchester, R. J., Capra, J. D. 1973. Cross-idiotypic specificities amongmonoclonal IgM protein with anti-v-globulin activity. J. Exp. Med. 137:331-39 61. Shoenfeld,Y., Isenberg, D. A., Rauch, J., Nadaio, M. P., Stollar, B. D., Schwartz,R. S. 1983.Idiotypic crossreactions of monoclonallupus antibodies. J. Exp. Med.158:178-85 62. Diamond, B., Solomon, G. 1983. A monoclonalantibody that recognizes anti-DNAantibodies in patients with systemic lupus. In ImmuneNetworks, ed. C. Bona, H. Kohler, 418:379-87 Ann.N.Y. Acad. Sci. 63: Davidson, A., Preud’homme,J. L., Solomon,A., Chang,M. D. Y., Beede, S., Diamond, B. 1987.Idiotypic analysis of myelomaproteins: anti-DNA activity of monoclonal immunoglobulins bearing an SLEidiotype is more commonin IgG than IgM antibodies. J. Immunol.138:1515-18 64. Rauch,J., Murphy,E., Roths, J. B., Stollar, D., Schwartz,R. S. 1982. A high frequency idiotypic marker of anti-DNAautoantibodies in MRL/Ipr

mice. J. Immunol.129:236-41 65. Gavalchin,J., Datta, S. K. 1987. The NZBx SWR model of lupus nephritis. II. Autoantibodiesdeposited in renal lesions showa distinctive andrestricted idiotypic diversity, d. Immunol.138: 138-48 66. Pisetsky,D. S., Lerner,E. A.1982.Idiotypic analysis of a monoclonal anti-Sin antibody. J. Immunol.129:1489-91 67. Cohen,P. L., Eisenberg, R. A. 1982. Antiidiotypic antibodies to the Coombsantibody in NZBF1 mice. J. Exp. Med. 156:173-80 68. McCoy,J. P., Michaelson, H. H., Bigazzi, P. E. 1983. Antiidiotypic immunity and autoimmunity. III. Investigations in humanautoimmune thyroiditis. L(fe Sci. 32:109-18 69. Dwyer,D. S., Bradley,R. J., Urquhart, C. K., Kearney,J. F. 1983. Naturally occurring antiidiotypic antibodies in myastheniagravis patients. Nature301: 611-14 70. Monestier, M., Manheimer-Lory,A., Bellon,B., Painter,C., Dang,H., Talal, N., Zanetti, M., Schwartz,R., Pisetsky, D., Kuppers,R., Rose,N., Brochier,J., Klareskog,L., Holmdahl,R., Erlanger, B., Alt, F., Bona,C. 1986.Sharedidiotopes and restricted VHgenes characterize murineautoantibodiesof various specificities. J. Clin. lnvest. 78: 75359 71. Penhale,W.J., Irvine, W.J., Inglis, J. R., Farmer, A. 1986. Thyroiditis in T cell depleted rats: Suppressionof the autoallergic responseby reconstitution with normallymphoidcells. Clin. Exp. ImmunoL25:6-16 72. Aron, R. 1981. Experimentalallergic encephalomyelitissusceptibility and suppression. Immunol,Rev. 55:5-30 73. Moore,J. S., Calkins, C. E. 1985. In vitro regulation of the pathogenic autoantibodyresponse of NewZealand Black mice. I. Loss with age of suppressiveactivity in T cell population.J. Immunol. 135:3838-44 74. Bona, C., Paul, W.E. 1979. Cellular basis of expressionof idiotypes.I. Suppressor cells specific for MOPC460 idiotype regnlate the expression of cells secretinganti-trinitrophenylantibodies bearing 460 idiotype. J. Exp. Med. 144:592-600 75. Liebermann,R., Bona, C., Chien, C. C., Stein, K. E., Paul, W.E. 1979. Genetic and cellular regulation of the expressionof specific antibodyidiotypes in the antipolyfructosan immune response.Ann.Immunol.(Inst. Pasteur) 130C:247-62

Annual Reviews

Annu. Rev. Immunol. 1988.6:327-358. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

V GENESOF AUTOANTIBODIES 76. Bona, C., Hooghe,R., Cazenave, P.A., LeGuern, C., Paul, W. E. 1979. Cellular basis of regulation of expression of idiotypes. II. Immunity to anti-MOPC460 idiotype antibodies increases the level of anti-trinitrophenyl antibodies bearing 460 idiotypes. J. Exp. Med.149:815-23 77. Kelsoe, G., Reth, M., Rajewsky,K. 1981.Controlof idiotype expressionby monoclonalantiidiotype and idiotype bearing antibodies. Eur. J. Immunol. 11:418-23 78. Hiernaux,J., Bona,C., Baker,P. 1981. Neonatal treatment with low doses of antiidiotypic antibody leads to the expression of a silent clone. J. Exp. Med. 153:1004-8 79. Lefvert, A. K. 1987.Antiidiotypeantibodies in myastheniagravis. In BiologicalPropertiesof Antioidiotype Antibodies,ed. C. Bona.Florida: CRIPress. In press 80. Bona,C. A., Heber-Katz,E., Paul, W. E. 1981. Idiotype antiidiotype regulation. I. Immunizationwith a levanbinding myelomaprotein leads to the appearanceof autoanti(antiidiotype) antibodies and to the activation of silent clone. J. Exp. Med.153:951-67 81. Zanetti, M., Rogers, J., Katz, A. H. 1984. Induction of autoantibodies to thyroglobulin by antiidiotypic antibodies. J. Immunol.133:240-43 82. Essani, K., Srinivasappa, J., McClintock, P. R., Prabhakar,B. S., Notkins, A. L. 1986. Multiple organ-reactive IgG antibody induced by an antiidiotypic antibody to a humanmonoclonal IgMautoantibody.J. Exp. Med. 163:1355-60 83. Sege, K., Paterson, P. A. 1978. Useof antiidiotypic antibodiesas cell-surface receptor probes. Proc.Natl. Acad.Sci. USA75:2443-47 84. Marion,R., Elias, D., deLongh,B. M., Bruining, G. J., van Rood, J. J., Schechter, Y., Cohen, I. R. 1983. Autoantibodiesto the insulin receptor in juvenileonset insulin dependentdiabetes. Nature 303:817-18 85. Farid, N. R., Briones-Urbina, R., Nazruil-Islam, M. 1982. Biological activity of anti-thyrotrophin antiidiotypic antibody.J. Cell Biochem.19: 305-13 86. Fong,S., Gilbertson,T. A., Carson,D. A. 1983. Theinternal imageof IgGin cross-reactiveantiidiotypic antibodies against humanrheumatoidfactors. J. lmmunol. 131:719-24 87. Schreiber, A. B., Courand, P. O., Andre, C., Vray, B., Strossberg, A.

357

1980. Anti-alprenelol anti-Id antibodies bind to fl-adrenergic receptors and modulatecatecholamine-sensitive adenylate cyclase. Proc. Natl. Acad. Sci. USA77:7385-89 88. Fraser, C. M., Venter, J. G. 1980. Regulation of fl-adrenergic receptor density in the control of adrenergic responsiveness. In Membrane,Receptors and the ImmuneResponse,ed. E. P. Cohen,H. Kohler, pp. 127-44. New York: Liss 89. Bona,C. A., Yang,C.-Y., Kohler, H., Monestier, M. 1986. Epibody: the imageof the networkcreatedby a single antibody. Immunol.Rev. 90:115-28 90. Geltner, D., Franklin, E. C., Fragione, B. 1980. Antiidiotypicactivity in the IgM fractions125:1539-36 of mixedcryoglobulins. J. Immunol. 91. Renversez,J. C., Roussel,S., Vallee, M. J., Brighouse, G., Lambert,P. Y. 1984.Bindingactivity of cryoglobulins. Blood Trans. Immunohematol. 27: 73755 92. Holmdahl,R., Nordling, C., Rubin, K., Tarkowski,A., Klareskog,L. 1986. Generation of monoclonalrheumatoid factors after immunizationwith collagen II-anti-collagen. II immune complexes. Ananti-idiotypic antibodyto anti-collagen II is also a rheumatoid factor. Scand. J. lmmunol.25:197-203 93. Bottazzo, G. F., Todd, I., Mirakiaw, R., Belfiore, A., Pujol-Borrel,R. 1986. Organ-specific autoimmunity:a 1986 overview. Immunol.Rev. 94:137-69 94. Harley,J. B., Reichlin, M., Arnett, F. C., Alexander,E. L., Bias, W.B., Provost, T. T. 1986. Geneinteraction at HLA-DQ2 enhances autoantibody production in primary Sjogren’s syndrome. Science 232:1145-47 95. Zabriskie, J. B., Hsu,K. C., Segal, B. C. 1970. Heart reactive antibody associated with rheumaticfever: characterization and diagnostic significmace.Clin. Exp. Immunol.7:147-54 96. Husby,G., van de Rijn, I., Zabriskie, J. B., Abdin, Z. H., Williams, R. C. 1976. Antibodiesreacting with cytoplasm of subthalamic and caudate nuclei neurons in chorea and acute rheumatic fever. J. Exp. Med. 144: 1094-99 97. Zabriskie, J. B., Freimer,E. H. 1966. An immunological relationship between the group A streptococcus and mammalian muscle. J. Exp. Med. 124: 661-72 98. Williams, C. L., Lennon,V. A. 1986. ThymicB lymphocyteclones from patients with myastheniagravis secrete

Annual Reviews

Annu. Rev. Immunol. 1988.6:327-358. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

358

BONh

monodonalstriational autoantibodies reactingwithmyosin,~ actinin or actin. J. Exp. Meal. 164:1043-59 99. Kabat,E. A., Nickerson,K. G., Liao, J., Grossbard, L., Osserman,E. F., Glickman,E., Chess, L., Robbins, J. B., Sehneerson,R., Yasig, Y. 1986. A human monodonal macroglobulin withspecificity for ~(2-8)linked polyN-acetylneuraminicacid, the capsular polysaceharide of group B meningococeiand Escherichiacoli K~which crossreacts with polynucleotides and with denaturated DNA.3. Exp. Med. 164:642-56 I00. Naparst¢k,Y., Duggen,D., Schattner, A., Madaio,M. P., Goni, F., Fragione, B., Stollar, D., Kabat,E. A., Schwartz, R. S. 1985. Immunochemicalsimilarities between monoclonal antibacterial Waldenstrom’smacroglobulins and monoclonalanti-DNAlupus autoantibodies. J. Exp. Med. 161: 1525-38 101. Naparstek, Y., Andre-Schwartz,J., Manser,T., Wysocki,L. J., Breitman, L., Stollar, B. D., Gefter,M., Schwartz, R. S. 1986. A single germlineVngene segment of normal A/J mice encodes autoantibodies characteristic of systemic lupus erythematosus. Y. Exp. Med. 164:614-26 102. Kaline, S., Dvilansky,A., Estok, L., Nathan,I., Salotov,Z., Sarov,I. 1981. Detection of anti-platelet antibodies in patients with idiopathic thrombocytopenicpurpuraand in patients with rubella and herpes group viral infections. Clin. Exp. ImmunoL 44:49-56 103. Stollar, B. D., Schwartz,R. S. 1986. Monoclonalanti-DNAantibodies; the targets and origins of SLEautoantibodies. Ann.N.Y. Acad.Sci. 475: 19299 104. Manheimer-Lory,A. J., Bellon, B., Bona, C. A. 1987. Rheumatoidfactors and aging. In Aging and the Immune Response,ed. E. A. Goidl, pp. 329-44. NewYork, Basel: M. Dekker 105. Notkins, A. L., Takashi, O., Prabhakar, B. 1984. Virus-induced autoimmunity. In Concepts in Viral Pathogenesis,ed. A. L. Notkins,M.B. A. Oldstone, pp. 210-15. NewYork: Springer-Verlag 106. Coutelier,J. P., vander Logt,J. J. M.,

Heessen, F. W.A., Warmier,G., van Snick, J. 1986. Rheumatoid factor production in 129/Svmice: involvementof an intestinal infectious agent. J. lmmunoL 137:337-40 107. Shores, E. W., Eisenberg, R. A., Cohen,P. L. 1986.Roleof the Smantigen in the generation of anti-Sm autoantibodies in the SLE-proneMRL mouse. J. Immunol. 136:3662~7 108. Logtenberg, T., Kroon, A., GmeligMeyling,F. H. J., Ballieux,R. G.1986. Productionof anti-thyroglobulin antibody by blood lymphocytesfrom patients with autoimmunethyroiditis, induced by the insolubilized autoantigen. J. lmmunoL136:1236-40 109. Janke, U., Fischer, E. H., Alvord,E. C. 1985. Sequencehomologybetween certain viral proteins and proteins related to encephalomyelitisand neuritis. Science229:282-89 1 I0. Dighiero, G., Lymberi,P., Holmberg, D., Lundquist,I., Coutinho,A., Avrameas,S. 1985. Highfrequencyof natural autoantibodies in normal newborn mice. J. Immunol.134:765-71 111. Holmberg, D., Wennerstrom, G., Andrade, L., Coutinho, A. 1986. The high idiotypic connectivity of natural newbornantibodies is not found in adult mitogen-reactive B cell repertoires. Eur. J. ImmunoL 16:82-87 112. Notkins, L., Prabhakar, B. S. 1986. Natural autoantibodiesthat react with multiple organs: basis for reactivity. Ann. N.Y. AcadSci. 475:123-25 113. Monestier, M., Bona, C. 1987. Antibodies possessing multiple antigen specificities and exhibiting extensive idiotypiccross-reactivity. Intern. Rev. lmmunol.In press 114. Holmberg,D. 1987. Highconnectivity, natural antibodies preferentially use 7183and QVPC52 Vnfamilies. Eur. J. Immunol. 17:399-405 115. Dildrope, R., Krawinkel,V., Winter, E., Rajewsky, K. 1985. Vn gene expression in murine lipopolysaccharide blasts distribute over nine knownVngene groups and maybe random.Eur. J. Immunol.15:1154-56 116. Cohen,I. R., Cooke,A. 1986. Natural autoantibodies might prevent autoimmunediseases, lmmunol.Today 7: 363~54

Annu. Rev. Immunol. 1988.6:327-358. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Annu. Rev. Immunol. 1988.6:327-358. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Annual Reviews www.annualreviews.org/aronline

Annu. Rev. Immunol. 1988.6:359-380. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Ann. Rev. Immunol.1988.6: 339~80

ROLE OF THE MAJOR HISTOCOMPATIBILITY COMPLEXCLASS I ANTIGENS IN TUMOR GROWTH AND 1 METASTASIS Kenichi Tanaka, Takayuki and Gilbert Jay

Yoshioka,

Charles

Bieberich,

Laboratory of Molecular Virology, National Cancer Institute, Maryland 20892

Bethesda,

INTRODUCTION The major histocompatibility complex (MHC)of the mouse encodes three subfamilies of class I genes that encode membrane-boundor secreted glycoproteins involved in immuneregulation and functions (1, 2). While those class I genes that map to the Qa and Tla subregions of the MHC encodetissue-restricted differentiation antigens of yet unknownfunctions, those that map to the H-2 subregion encode widely distributed surface antigens whosefunctions are rapidly being defined at the molecular level (2, 3). Initially identified on the basis of their participation in graft rejection, the H-2 class I antigens differ from their structurally related Qa and Tla class I molecules in that they are highly polymorphic. For some of the H2 class I genes, the numberof alleles within the mousepopulation has been estimated to be greater than 100 (1). There is increasing evidence for intergenic exchanges of DNAbetween different class I genes within the genome, by a mechanismreferred to as gene conversion, which is responsible for the generation of such diversity (4). The combination of the ~ The USGovernmenthas the right to retain a nonexclusive royalty-free license in and to any copyright covering this paper.

359

Annual Reviews

Annu. Rev. Immunol. 1988.6:359-380. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

360

TANAKA ET AL

multigenicnature and the polymorphiccharacteristic of these H-2class I antigens makesit virtually impossible to have a perfect tissue match betweenindividualsandto avoid graft rejection in transplantationstudies. This latter phenomenon suggests an immunologic function for this unique family of geneproducts. TheH-2class I antigens, whichinclude the K, D, and L molecules,are classical transmembrane glycoproteins (5). A stretch of about 25 hydrophobicaminoacids withinthe moleculeis responsiblefor its insertion into the cell membrane. Theuniquespecificity of these class I antigens resides in entirety on the extracellular portion of each molecule,the region exhibiting the polymorphicsubstitutions. Single aminoacid replacementsat specific sites within this extracellular domainwill suffice to inducegraft rejection(5). One of the most significant concepts in modemimmunologycomes fromthe suggestionthat T cells recognize"foreign" antigens present on surfaces of aberrant cells only in the context of "self" antigens encoded for by the MHC (6, 7). Specifically, the H-2 class I antigens appear direct the recognitionof neoplastic and virus-infectedcells by cytotoxicT lymphocytes(CTL). ROLE OF MHC MOLECULES RECOGNITION BY T CELLS

IN

ANTIGEN

BothT and B ceils recognizeforeign antigens via cell surface receptors. UnlikeB cells, whichrecognizefree antigens, T cells recognizecell-surface antigens almost exclusively in association with moleculesencodedby the MHC.This phenomenon is called MHC restriction (6, 7). Differentsubsets of matureT cells are restricted by different classes of - cells (in most cases, cytotoxic MHC molecules. While Lyt2+/L3T4 lymphocytes)recognizeantigens in the context of MHC class I molecules, ÷ cells (usually helper T lymphoeytes)recognize antigens Lyt2-/L3T4 association with MHC class II gene products (8, 9). MHC restriction, well as specific antigen recognition, are largely mediatedthroughthe Tcell antigen receptor (TCR)(10-13) and are thought to be acquired during the intrathymic phase of T-celt development (14-17). Thepredominantclass of TCRis composed of a glycosylated, disulfidelinked heterodimerwith a combinedapparent molect[lar weight of 80-90 kd. The heterodimerconsists of a 48-54 kd acidic ~-chain and a 40-44 kd neutral to basic/~-chain(18, 19), each chain possessingvariable andconstant domainssimilar to those found in immunoglobulinmolecules. A second TCRheterodimer, composedof a V- and a di-chain, has also been identified on the surfaces of peripheral T cells and of a thymocytesubset

Annual Reviews

Annu. Rev. Immunol. 1988.6:359-380. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

CLASS I ANTIGENS AND TUMOR GROWTH

361

in both mice and humans(20-25). The TCRheterodimers are associated on the cell surfacewith several other molecules,collectively called the T3 complex, whichmaybe involved in signal transduction. The murine T3 complexincludes two glycoproteins of 25 kd and 21 kd, and two nonglycosylatedpolypeptidesof 26 kd and16 kd (26, 27). Basedupon the unique requirements for T cell antigen recognition, several experimentalmodelshave been proposedto provide a conceptual frameworkfor further studies (28-33). The"one-receptor" modelproposes that T cells recognize both antigen and MHC either as a combinational determinantor as two individual determinantsrecognizedby distinct binding sites on the same TCRmolecule. In the "two-receptor" model, one receptor recognizes the antigenic determinant(s), and a second receptor binds the polymorphicsite(s) of the MHC molecule. Morerecently, "one-and-a-half receptor" modelhas been proposed, whereone chain is shared betweentwo distinct complexes(33). Whilethe complexity of the TCRhas so far eluded precise molecular definition, recent findings appearto favor a single-receptor recognizing either one combinationalor two separated determinants. Antigen recognition is directly influenced by the MHC haplotype of the antigen-presenting cell for both class I- andclass II-restricted T cells (34-36), and antigen specificity and MHC restriction do not segregate independently by fusion of two T cell hybrodimaswith different specificities (37). addition, transfection of functional TCRgenes from a MHC-restricted CTLclone into another CTLclone of a different specificity endowedthe recipient cell withthe specificityof the donorcell (13). In a recent elaboration of the one-receptor model, a single TCRwas proposed to bind both the antigenic determinant and the "non-polymorphic" region of the MHCclass I molecule (31). Whenthe MHC moleculeand the antigen are broughttogether on the cell surface by the TCR,the stability of the bondto the TCRwill dependon the interaction between the MHC molecule and the antigen. The polymorphicregion of the MHC moleculecan directly influence its interaction with the antigen. If the repulsive forces between the MHC polymorphicregion and the antigen are too great, the TCRwill not be able to stably bind the two moleculessimultaneouslyon the cell surface. This modelnot only accounts for MHC restriction, in whichself moleculeshave to be presented on the cell surfacebeforeT cells can recognizea foreign antigen, it also explains whyan extraordinarily large numberof T cells respond to foreign MHC molecules.In the latter case, both bindingsites of the TCRwill be occupied by the sameforeign MHC molecule, and there will be no repulsive interaction. In the absenceof a repulsive force, foreignantigen bindingsites of lower affinity will give the complexan above-thresholdenergyand will

Annual Reviews

Annu. Rev. Immunol. 1988.6:359-380. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

362

TANAKA ET AL

result in stimulation of a muchlarger proportion of T cells. With this model, the TCRcontains a binding site for the nonpolymorphicregion of the MHCmolecule as a prerequisite (31). Experimental support has recently been obtained showing that antigenic proteins are not presented to helper T cells as intact molecules. Rather, they are first broken downto peptides in the antigen-presenting cells. The resulting immunogenicpeptides then bind directly to the MHCclass II molecules (38-42). Thus, the MHCclass II molecules behave muchlike receptor that has a single binding site for peptides derived from foreign antigens. This meansthat T cells recognize foreign peptides after they have bound to appropriate class II molecules and that the role of the class II molecules is to hold the peptides in a favorable conformation for T cell recognition. In addition, these studies also demonstrated that the absence of binding of a peptide to class II molecules can be correlated with immune unresponsiveness for certain antigen-strain combinations, and the absence ofT cells capable of recognizing the boundantigen can account for immune tolerance (43). These studies have addressed specifically the function of the MHC class II moleculesthat participate in interactions involving helper T cells. Generally, the MHC class I molecules are the restriction elements for CTL, and it will be interesting to see if class I molecules also function in the same way as class II molecules. Several recent studies have suggested that CTLalso preferentially recognize degraded fragments of viral proteins and allogeneic class I antigens in the context of self class I molecules (44, 45).

INVOLVEMENT OF CLASS I ANTIGENSIN THE PRESENTATION OF TUMORCELLS Tumor-specific transplantation antigens appear to display extensive "polymorphisms," in that each independently induced tumor seems to possess its ownspecific antigen (46-48). This remains true even when the tumors are induced in the same mouse strain, from the same organ system, and by the same chemical carcinogen. However, the host’s immunesystem can recognize these specific antigens and develop different anti-tumor responses. In particular, T cell-mediated immunity appears to have a significant role in eradicating tumorsin vivo (49, 50). BecauseT cells have a dual specificity for both tumor antigens and self MHCmolecules, the majority of tumor-specific CTLresponses detected in vitally and chemically induced tumormodelsare class I restricted (49). For example, CTLwith specificity for the simian virus 40 (SV40)-specific antigen can be generated in mice with SV40-transformed cells. These

Annual Reviews

Annu. Rev. Immunol. 1988.6:359-380. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

CLASS I ANTIGENS

AND TUMOR GROWTI-I

363

CTLare MHC-restricted(50), and their ability to respondis regulated specifically by the Kand D gene products(51-53). Furthermore,evidence for the involvementof SV40antigen-specific CTLin immune resistance to tumor growth comesfrom the observation that following adaptation to rapid in vivo growthin nude mice, SV40-transformed cells were found to growas tumorsonly in low, but not in high, CTLresponderstrains (54). In addition, adapted tumorcells no longer expressed class I antigens. Thesestudies demonstratedthe essential role of class I moleculesin the presentation of tumor-specificantigens to the host’s immune system. Natural killer (NK)cells are lymphoideffector cells possessingspontaneous cytolytic activity against a variety of tumorcells (55). Thefact that NKcells preexist at high frequencyand require no lengthyactivation, and the observationthat differentiated and metastatic tumorcells often havea decreasedsensitivity to NKactivity, haveled to the hypothesisthat NKcells maybe involved in anti-tumor surveillance especially in early stages of tumor development(56). The ontogenyof NKcells and their target recognition mechanisms remain unclear. Recently, Karre et al haveproposeda modelfor antigen recognition in whichNKcells might.detect the "absence of the expected (MHC)" rather than the "presenceof the unexpected(antigen)" (57, 58). In this model, class I moleculesprovide a self signal that will inhibit triggering and subsequentdelivery of lytic events. Severalobservationsdemonstratingan inverse correlation betweenMHC expression and NKsensitivity seemto support this hypothesis(59-64). CONTROL OF EXPRESSION

OF CLASS I

ANTIGENS

Immunocompetent syngeneic hosts can respond to transformed cells by different effector mechanisms responsible for limiting tumorgrowthand metastasis.At least in certain cases, as discussedabove,antigens expressed on tumor cells are presented to T cells in association with self MHC molecules.In particular, the class I moleculeshavebeensuggestedto be essential for presentingantigens to Lyt2+ CTLand, thus, play an essential role in the immuneelimination of certain tumors. Therefore, any perturbation in the expression of class I genes in transformed cells may profoundlyaffect the host’s anti-tumoractivities (65). Alterations of class I antigens that have beenobservedon tumorcells can be groupedinto two categories: (i) the generation of novel class antigens, and(ii) the suppressionor loss of specific class I antigens. While these phenomena are not observed in all tumor systems, alterations of class I antigens in association with the development of tumorsmaybe one of several decisive factors that determinethe course of certain malig-

Annual Reviews

Annu. Rev. Immunol. 1988.6:359-380. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

364

TANAKA ET AL

nancies. Welater discuss the evidencethat tumor-associatedchangesof class I antigens are responsiblefor the tumorigenicphenotypeof cells. Recognizingthe importance of the control of expression of class I antigens in immune processes, manyinvestigators havefocusedtheir attention on understandinghowclass I genes are regulated in both normaland malignantcells. Bystudyingfactors that can affect class I genetranscription, mRNA processing,or antigen presentationon the cell-surface, it will be possible to design strategies for restoring the expression of class I antigens in tumorcells. Suchmodalities could have tremendousclinical significance. Theregulation of class I geneexpressionhas beenextensively studied (66). In the mouse,transcription of class I genesis first detectable in day 9 embryos,and cell-surface expressionof class I antigens is manifestedby the mid-somitestage on day 10 (67). Earlier expressioncan be induced treatment of dissociated embryoniccells with interferon (IFN)in vitro. Developmentalactivation of the K gene has been followed in a mouse embryonalcarcinoma(EC) cell line (68). ECcells can be induced differentiate and present developmentalanalogies with early embryonic cells. Treatmentof ECcells with retinoic acid inducesexpressionof class I antigens by a mechanismthat appears to involve an increase in DNA methylation (68). This observation is in contrast to the manyexamples wheretranscriptional activity of genesappears to be inversely correlated with the extent of DNA methylation (69). In the adult mouse, class I antigens are often thought of as being ubiquitousin terms of tissue distribution. Early experimentsdemonstrated that class I antisera could immunoprecipitate material from virtually all tissues, althoughin varying amounts(70). Morerecently, this observation has been called into question. Immunohistochemical staining of tissue sections has demonstratedthat cells in the renal tubules (71, 72) and pancreaticacinar cells are both negativefor class I antigens(73). However, it should be noted that studyingclass I expressionin tissue sections is a difficult task. Fixation often renders these antigens unreactiveto specific antibodies, and frozen sections characteristically lack well-defined morphology. It is likely that while mostcells expressclass I antigens, the levels of expressionon the cell surface can be highly variable, evenwithin a population of cells of the samelineage. Furthermore,in cells that are negative or marginalfor class I antigens, expressioncan be dramaticallyincreased by treatment with physiologicand pharmacologic agents (66). In cells that alreadyexpresssignificant levels of these antigens, expressioncan still be further up-regulatedby these sameagents. Themostwell-characterizedclass I modulatingagents are the interferons

Annual Reviews

Annu. Rev. Immunol. 1988.6:359-380. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

CLASS I ANTIGENS AND TUMOR GROWTH

365

(IFNs). Lindahl et al (74) were the first to describe the induction of class I antigens on mouselymphomacells in culture. These observations have been confirmed for manycell types, both as primary cultures or as established cell lines. While the relative increase in class I expression can be manyfold, cells that have a low constitutive level of expression generally respond to IFN treatment to a muchgreater degree than do cells with a high constitutive level. Both type I (~ and fl) and type II (~) IFNs capable of eliciting and increasing class I gene expression, although IFN~ appears to be more potent in certain cell types studies (75). The mechanism(s)whereby IFN acts to increase cell-surface expression of class I glycoproteins are beginning to be elucidated at the molecular level. It appears that transcriptional control plays a major role in the IFN response since class I mRNA accumulation is nearly always increased in IFN-treated cells. However,post-transcriptional regulation mayalso be important (76). Initial experiments to delineate the cis-acting DNA sequences required for IFN induction yielded surprising results. Yoshie et al (77) demonstrated that mousecells transfected with promoterless human class I genes still expressed IFN-inducible HLAantigens. More recent analysis of the 5’ region of several HLAclass I genes has led to the identification of 30-bp interferon response sequence (IRS) (78). sequence was also found to be involved in the regulation of a mouseclass I gene (79, 80). In conjunction with a specific enhancer sequence, the class I IRS can confer IFN inducibility to heterologous genes (79). It is now believed that cis-acting DNAsequences both 5’ and 3’ to the transcription initiation site are required to achieve full induction (80)~ Despite these advances in the characterization of the IFNresponse, it is still not clear whether IFN acts directly to induce class I transcription or whether other mediators are involved. Autocrine regulation of class I expression by production of IFN has been demonstratedin vitro (81). The relevance of this observation to class I modulation in vivo needs to be established, especially in light of the observation that regions surrounding inflammatory infiltrates in muscle disease have an increased expression of class I molecules (82). Perhaps cells under stress increase their level of class I expression by producing IFN. Tumornecrosis factor also appears to induce the expression of class I genes in cultured cells (83), possibly through a mechanismthat involves the induction of IFN (84). The anti-inflammatory drug, indomethacin, mayindirectly increase class I expression by inhibiting prostaglandins (66). Administration of lipopolysaccharide also appears to increase class I on mouserenal tubules. In addition, two pharmacologicagents, calcitriol (85) and sodiumbutyrate (86), have been shownto up-regulate class I antigens

Annual Reviews 366

TANAKA ET AL

on tumorcells in vitro. Treatmentof class I-deficient BL6melanoma cells with N-methyl-N’-nitronitrosoguanidinehas also been shownto increase dramaticallythe level of class I moleculeson these cells and to increase their immunogenicity (87).

Annu. Rev. Immunol. 1988.6:359-380. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

QUALITATIVE CHANGESOF CLASS I ANTIGENS ON TUMOR CELLS Manyreports in the literature demonstratetumor-specific expression of MHC antigens alien to the mousestrain from whichthe tumorwas derived (88-90). The presence of an "alien" MHC antigen in a tumor cell line is generally established by using alloantisera specific to different MHC haplotypes.Initially, difficulties wereencountered in establishingthe true nature of these aberrant moleculesbecauseof potential cell contamination, transient expressionof somespecificities (91), lowlevel of representation on the tumorcell surface(89), andpresenceof antiviral antibodiesin many of the alloantisera used. However, in at least several tumorsystems,further studies have been carried out using monoclonalantibodies and immunological assays, and these have confirmedthe initial observations. In someof these cases, aberrant moleculeshave also been characterized by biochemical means. Schmidt& Festenstein (92) have detected an aberrant D°-like allospecificity in the K36.16thymoma cell line fromAKR (H-2k) mice. Isozyme markersof K36.16cells wereidentical to those of the AKR strain, thereby confirmingthe origin of these tumorcells. Whilethe initial study was performedusing alloantisera, the observationhas recently been confirmed by the use of monoclonalantibodies (93). The finding that K36.16cells werespecifically lysed by anti-D° CTL(89) further suggeststhat the alien specificity is D°-like.Trypticpeptideanalysisof the aberrantclass I antigen o_ obtained by immunoprecipitationrevealed the presence not only of D specific peptides but also of a numberof extra components(89, 92). addition, a novel restriction enzymefragmenthas been identified using specific class I DNA probes in K36.16DNA whichis absent from parental AKRDNA(94). Aberrant class I moleculeshave also been detected on an ultraviolet radiation-induced fibrosarcoma 1591. Schreiber and collaborators have extensively analyzedthis C3H-derived tumorline to identify novel tumorspecific antigens (95). Monoclonalantibodies generated against the 1591 fibrosarcomabound specifically to these tumor cells but not to other syngeneic fibrosarcomas, nor to embryonicor adult normal syngeneic cells. However,these monoclonalantibodies cross-reacted with certain allogeneic class I antigens. Furthermore,anti-Ld monoclonalantibodies

Annual Reviews

Annu. Rev. Immunol. 1988.6:359-380. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

CLASS

I

ANTIGENS

AND TUMOR GROWTH

367

and anti~L d CTLlines cross-reacted with the 1591 tumor but not with normal C3Hcells (96). Tryptic peptide mappingstudies have led to the suggestion that the novel antigens are similar to but distinct from class I products normally expressed by C3Hmice, and that one of these antigens shares Ld-like epitopes (97). Goodenowand coworkers have subsequently cloned the genes that encode these novel antigens and have demonstrated that the 1591 tumor expresses, in addition to a normal complementof class I antigens, at least three novel class I molecules not normally expressed in C3Hmice (98). Twoof these genes are highly homologous to the Ld gene from BALB/c mice, and a third is quite homologousto the Kk gene throughout its 3’ region (99). Furthermore, analysis of fibroblastic lines transfected with each of the three novel class I genes indicates that these sequences encode products recognized by the same monoclonal antibodies and CTLclones that are reactive with the original 1591tumor (98, 99). These studies have led to speculations regarding the mechanism(s) responsible for generating novel class I sequences in tumor cells. Since these class I genes contain unique restriction fragments not detectable in the genomeof the mousestrains from which the respective tumors were derived, they must have been generated by multiple recombination events amongendogenousclass I genes and must have occurred during the course of tumorigenesis. For example, the C3Hf-derived lung carcinoma line (LT85) is rapidly rejected upon transplantation into parental C3Hfmice (100). Biochemical analysis of the Kk-like molecule expressed on this tumor has led to the suggestion that the gene which encodes this product was generated by a mutational event, such as a small sequence exchange, at the Kk locus (101). The same genetic mechanism(s)responsible for generating the enormous diversity observed between MHCantigens from different mousestrains maywell be operative in deriving alien specificities in tumors.

QUANTITATIVE CHANGESOF CLASS I ANTIGENS ON TUMORCELLS Viral infection of cells often leads to the appearance of virus-specific antigens on the cell surface. Theseviral antigens are readily exposedto the immunesystem of the host and are subject to elimination by class I restricted CTL(6, 7). As a means of escape from immunedetection, many viruses have evolved strategies for reducing the level of class I antigen expression on cells they infect. Such down-regulation of class I molecules may be involved in the development of leukemias caused by several murine retroviruses. Genetic control of resistance to neoplasia induced by retroviruses was

Annual Reviews

Annu. Rev. Immunol. 1988.6:359-380. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

368

TANAKA ET AL

first demonstratedin mice by Lilly (102). Theresistance locus for the Friend murine leukemia virus (F-MuLV)was shownto mapto the H-2D region of the MHC (103). The relationship between resistance to viral oncogenesis and the host’s ability to mounta CTLresponse has also been well documented(104, 105). AKRmice have a high incidence spontaneousleukemias.Schmidtet al (106) first observeda selective loss of the expressionof the Kk antigen on one such leukemia.That observation has been extendedto many,but not all, independentlyarising AKRleukemias (107). Levels of the k antigen i n AKR leukemic cells a ppear normaland mayeven be elevated in somecases. In addition, it has been demonstrated that the reductionin the Kk antigen is correlated with resistance in vitro to killing by CTL(108). Together,these observationsprompted the suggestionthat the Kk antigen, but not the Dk antigen, is capable of serving as a restriction element for AKRMuLV-specific CTL. Oneparticular AKR tumorcell line, designatedK36.16,has been studied in detail. Thesecells do not expressthe Kk antigenon the cell surface, are resistant to killing by AKR-MuLV CTLin vitro, and always form tumors in immunocompetent AKRmice (108). Hui et al (109) used mediatedgenetransfer to introduce a cloned Kk gene to rescue the class -I-deficient phenotypeof K36.16cells. Varioussubclonesof the Kk-trans fected 1(36.16cells were then tested for their ability to formtumorsin AKRand AKRx BALB/cF1 mice. In contrast to the parental 1(36.16 cells, transfected clones expressing the Kk antigen were inefficient at inducing tumors in immunocompetent recipients. Furthermore, there appearedto be an inverse correlation betweenthe level of the Kk antigen and tumorigenicity amongthe transfectants. These data clearly demonstrated the biological relevanceof the Kk antigen in controlling the in vivo growth of tumors in the AKRmouse. Therole of the class I antigens in T cell-mediatedsurveillance of radiation leukemiavirus (RadLV)-transformed cells has also beenextensively studied by Merueloand coworkers (110-112). The H-2Dregion of the MHC has been implicated in resistance to RadLV-induced oncogenesis. In resistant but not susceptible strains, the level of the D antigen on thymocytes increases after intrathymic inoculation with RadLV.In addition, an inverse relationship betweenviral antigen expression and D antigen expression has been observed. In resistant strains, infected thymocytesshowhigh levels of cell-surface D antigen and low levels of viral antigen. Theconverseis true for susceptible strains; leukemogenesis in these mice is characterized by a complete lack of class I antigen expressionin the infected cells. Resistanceis apparentlycorrelated with a strong CTLresponse which mayeffectively eliminate the RadLV-transformedcells. The mechanism(s)involved in RadLV-induced modulation

Annual Reviews

Annu. Rev. Immunol. 1988.6:359-380. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

CLASS I

ANTIGENS AND TUMOR GROWTH

369

of class I expression are not clear; however,a recent report demonstrated increased DNAmethylation and DNArearrangement within the MHC from RadLV-transformed thymocytes (111). Viruses have been shown integrate near the MHC,and regulatory roles have been postulated (111, 112). The relationship betweenclass I antigen expression and tumor formation has been well characterized for cells transformed with different DNA viruses. Infection of cultured mouse cells with simian virus 40 (SV40) results in cellular transformation (54). However,these transformed cells are immunogenicand rarely form tumors in immunocompetentsyngeneic hosts. SV40tumor-specific, Kk-restricted CTLare believed to be responsible for clearing tumor cells from these animals. Repeated passage of SV40-induced tumors in immunocompromisedanimals has led to the isolation of cell variants that are highly tumorigenic even in immunocompetent syngeneic recipients (54). These immunoselectedcells, unlike their precursors, are unable to serve as targets for CTL-mediatedlysis in vitro and can escape destruction in SV40immuneanimals in vivo. Gooding (54) has comparedseveral highly tumorigenic SV40-transformedcell variants with their nontumorigenic progenitors to determine the cause of their escape from CTLkilling. She has established that tumorigenic cells consistently failed to express the Kk kclass I molecules. It appearedthat K deficient cells had a selective advantage, presumablyon the basis of their ability to escape anti-SV40 specific CTLkilling. These results strongly implicated CTLas a major factor involved in tumor rejection in this system. Rogers et al (113) have traced the defect in k expression i n t he immunoselected SV40-transformed cells to a rearrangement at or in the vicinity of the Kk gene. The role of SV40in inducing this DNArearrangementis not clear. The ability of humanadenoviruses to regulate the expression of host class I antigens has also been extensively studied. Humanadenoviruses are categorized as non-oncogenicor as oncogenic based on their ability to induce tumors in rodents (114). Regardless of their tumorigenic potential, all adenovirus serotypes seem to have the propensity to transform rodent cells in culture. Cells transformed by the non-oncogenicserotypes, including Ad2 and Ad5, are nontumorigenic in syngeneic immunocompetent hosts but can form tumors in immunosuppressedrecipients. In contrast, cells transformed by the oncogenic serotypes, including Adl2, are highly tumorigenic even in syngeneic immunocompetentrodents. Initial experiments to determine the basis for the difference in oncogenicity of the various serotypes have focused on the early la (Ela) region of Ad5 and Ad12. Schrier et al (115) reported a striking difference in the level of class I expression in baby rat kidney cells transformed with Ad5 Ela or Adl2

Annual Reviews

Annu. Rev. Immunol. 1988.6:359-380. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

370

TANAKA ET AL

Ela regions. Adl2 Ela-transfectants had drastically reduced amounts of class I antigen compared to AdS Ela-transfectants. Adl2 Ela-transfectants also showed reduced susceptibility to lysis by allogeneic CTL in vitro, prompting the suggestion that the oncogenicity of the Ad12transformed cells could be explained by their escape from immunesurveillance by CTL. Interestingly, cells transformed simultaneously with both AdSand Adl2 Ela regions do not have reduced levels of class I antigens (116). Apparently, the product of the AdSEla gene can prevent the Ad 12 Ela product from suppressing class I expression. The relationship between the Adl2-transformed, class I-deficient phenotype and tumorigenicity has been studied in the mousesystem (117, 118). Primary mouse kidney cells transfected with the Adl2 Ela region are highly oncogenic in immunocompetent syngeneic adult mice. However, transfection and expression of a single cloned class I gene in these cells was sufficient to abrogate their tumorigenicity. This observation lent strong support to the hypothesis that the oncogenicity of Ad12-transformed, class I-deficient cells was a result of their ability to escape immunedetection. Further support for this hypothesis came from experiments in which the level of class I antigen in Adl2-transformed cells was up-modulated by treatment with IFN prior to inoculation of a tumorigenic dose into immunocompetent animals. Hayashi et al (119) demonstrated that pretreatment of Adl2-transformed cells with IFN resulted in a derepression of class I genes and a concomitant reduction of tumorigenicity of these cells in adult mice. In vivo administration of IFN subsequent to a tumor-inducing inoculumof Adl2-transformed cells also was sufficient to abrogate tumorigenicity in this system completely. The Ad12Ela-mediated suppression of class I genes appears to operate at the mRNA level. Ad12-transformed cells have greatly reduced steadystate levels of H-2 class I transcripts. The observation that IFN treatment derepressed class I genes in Adl2-transformed cells (119, 120) indicates that the class I genes are not permanently inactivated by mutation or deletion, but instead retain their ability to be expressed under appropriate conditions of stimulation. A recent report has demonstrated that the rates of transcription of Adl2-suppressed class I genes are not significantly different from those in AdS-transformed cells (121). This observation suggests that Ad12 suppression involves a change in the post-transcriptional processing or stability of class I mRNA. The perturbation in class I antigen expression may not completely explain differences in tumorigenicity amongdifferent adenovirus serotypes. Resistance to lysis by NKcells has been reported for Adl2-transformants (122), and this maybe importantfor the failure of these cells to be rejected. It also appears that not all Adl2-transformed cell lines are capable of

Annual Reviews

Annu. Rev. Immunol. 1988.6:359-380. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

CLASS I

ANTIGENS AND TUMOR GROWTH

371

inducing tumors in immunocompetent hosts (120). Mallowet al (123) have failed to observe a correlation betweentumorigenicpotential and allogeneicT-cellkilling in vitro. Otherviruses also appearto modulateclass I expression.Cells infected with vesicular stomatitis virus (124) andHerpessimplexviruses (125) reducedlevels of MHC antigens. The mechanismsinvolved in these cases are unknown.Furthermore, non-oncogenicadenoviruses also appear capableof altering the level of surface class I antigensin cells they infect (126). Ad2producesa glycoprotein, designatedEl9, that binds to nascent class I moleculesin the endoplasmic reticulumand preventstheir transport to the cell surface(127,128).

IMMUNOMODULATION OF EXPERIMENTAL TUMORS In mice, modulationof class I expressionby transfection of cloned class I genesinto class I~leficient, virally transformedcells has clearly been demonstrated to be effective in rendering those cells immunogenic in immunocompetent syngeneichosts. The success of this approachhas also been elegantly demonstratedfor a class I-deficient, chemicallyinduced sarcoma(129). Wehaverecently observedsimilar results for a spontaneous melanoma(K. Tanaka, unpublishedresults). The fact that current gene transfer technologydoes not allow specific targetting of genesto tumor ceils in vivo, severelylimits the clinical implicationof this approach. At the present time, other modalities for augmentingthe immunogenicity of tumorcells mayprove moreuseful. Treatmentof Ad12-transformed cells with IFN and treatment of BL6melanomacells with Nmethyl-N’-nitronitrosoguanidinein vitro not only induced class I expression but also decreasedthe tumorigenicityof these cells in vivo (87, 119). Theseobservationsunderscorethe needto identify chemotherapeutic agents that can derepressendogenous class I genesin tumorcells in vivo. Augmenting the immunogenicityof tumorcells by transfection or derepression of class I genes could also be used to potentiate the immune systemto recognizeand reject isogenic tumorcells expressinglow levels of class I molecules. Marginal expression of class I antigens maybe sufficient to present the transformedcells, providedthe immunesystem can be potentiated toward a moreeffective recognition of these cells. Tanakaet al (118) have recently demonstratedthe feasibility of this approach. Mice preimmunized with a nontumorigenic dose of IFNtreated, Ad12-transformedcells were completelyprotected against a subsequent challenge with a tumorigenic dose of untreated tumor cells. Immunitycan also be achievedby transfection of a cloned class I genein

Annual Reviews

372

TANAKA ET AL

this experimentaltumorsystem(118), as well as in the previouslydescribed AKR(109) and methylcholanthrene (129) tumor models.

Annu. Rev. Immunol. 1988.6:359-380. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

PAUCITY TUMORS

OF CLASS I

ANTIGENS

IN

HUMAN

Expressionof class I antigens is clearly requisite for the immune recognition of aberrant cells. Perturbationin the level of cell surface class I antigens in tumorcells mayhave significant clinical implications. Many humantumors, particularly those of epithelial derivation, appear to express greatly reducedlevels of surface class I molecules.Theyinclude small cell lung carcinoma(130), basal cell carcinoma(131), squamous carcinoma(132), eccrine porocarcinoma(133), and neuroblastoma(134). Tumorcell lines deficient in class I moleculeshavealso beenderivedfrom several of the above tumors and from mammary carcinoma (135, 136), colorectal carcinoma(137, 138), melanoma(139), and choriocarcinoma (137, 140). In an analysis of 15 independent skin biopsies of humanbasal cell carcinomas,all sampleslacked HLA class I antigens and fl2-microglobulin as defined by immunohistochemical staining (131). In contrast, benign epidermal lesions, including keratocanthomas, seborrheic warts, epidermoidcysts, and pilaf cysts were not deficient in class I antigen expression. Immunohistochemical analysis of squamouscell carcinomas and premalignantepithelial lesions havedemonstrateda lack of fl2-microglobulinin these cells (132). Absenceof fl2-microglobulincorrelated perfeetly with both histopathologicdiagnosis and clinical behaviorin all of 15 eccrine porocarcinomas(133); deficient fl2-microglobulinexpression correlated with malignanthistology and clinical metastasis but not with benigntumors. Theabsenceof class I and/or fl2-microglobulinexpression is apparently correlated with the development of epithelial malignancies. Severalhumancell lines derivedfrompatients with small cell lung cancer werealso deficient in class I antigens andfl2-microglobulinwhenanalyzed for cell surface expression by immunoprecipitationor for mRNA accumulation (130). In contrast, lung cancer cell lines derived from adenocarcinoma, epidermoid, and mesotheliomacancers had normal levels of class I antigens.It is interestingto note that smallcell lungcancertypically exhibits more rapid growth and earlier metastasis than do the other lung malignancies;this suggests a loss of immune control. Smallcell lung cancer biopsies taken directly from patients and analyzed by immunohistochemicalmethodsalso had decreasedclass I expression, while other types of lung cancerdid not (130). This observationdemonstratedthat the

Annual Reviews

Annu. Rev. Immunol. 1988.6:359-380. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

CLASS

I

ANTIGENS

AND TUMOR GROWTH

373

decreasedclass I antigen expressionwasnot an artifact inducedby cell culture. Neuroblastomacell lines, as well as metastatic neuroblastomatumor cells from the bone marrow,have similarly been shownto have decreased levels of HLAclass I antigens by immunohistochemicaI staining and by immunoprecipitation (134). In addition to havingdecreasedclass I antigen expression, manyneuroblastomasand small cell lung cancers also show amplification of one of the membersof the myconcogenes(141,142). recent report has strongly implicated the product of the N-mycgene in down-regulationof class I geneexpressionin neuroblastomas (143). Using a rat neuroblastomamodelsystem, Bernardset al demonstratedthat overexpression of the N-mycgene resulted in the down-modulation of class I antigen expression that was reversible by IFNtreatment. The in vivo growthrate andmetastatic potential of these cells also increased. As more tumors are analyzed for class I and ~2-microglobulin expression,it is likely that evenmorecorrelations betweendeficient class I expression and malignancywill be made. In addition, it mayeven be possible to correlate decreasedexpressionwith the degreeof the malignant phenotype.

CONCLUSION Cancermaybe thought of as an immunological disorder that arises because certain "transformed"cells, endowed with the propensity to divide, have learned to evade detection by the immunesystem. Theprospect of intervention by "immunotherapy" dependsvery muchon our ability to either (i) render cancer cells morerecognizableby the immune system, or (ii) potentiate the immunesystem towards a more effective recognition of cancercells. There is nowdirect evidencethat the suppressionof the major histocompatibility complexclass I antigens are directly responsible for the escape of sometumorcells fromimmune detection. It has beenshownthat cancer cells can be madeimmunogeniceither by the expression of an exogenousclass I gene introduced by DNA-mediated gene transfer or by the derepression of endogenous class I geneswith interferon; these cells are efficiently rejected by the immune system.Evenmoreinteresting is the finding that the immunesystem can be potentiated to reject tumors by immunizationwith homologoustumor cells that have been manipulated to expressnormallevels of class I antigens. Since increasing numbersof humantumors have been found to have greatly reducedlevels of class I antigens, these findings suggesta direct route to immunotherapywhich involves debulking of the tumor mass,

Annual Reviews 374

TANAKAET AL

raising the level of class I antigens in a small numberof explantedtumor cells, and reimmunizing the host. The response to the sensitizing tumor cells mayfurther be augmented by the T cell growthfactor, interleukin-2. In the years ahead, there maybe increasing interest in such immunotherapy as a modalityfor the treatment of humancancers.

Annu. Rev. Immunol. 1988.6:359-380. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Literature Cited 1. Klein, J., Figueroa, F., Nagy,Z. A. 10. Kronenberg,M., Siu, G., Hood,L. E., 1983. Genetics of the major histoShastri, N. 1986. Themolecular gencompatibility complex:the final act. etics of the T-cell antigenreceptor and Ann. Rev. lmmunol.1:119-42 T-cell antigen recognition. Ann.Rev. 2. Hood,L., Steinmetz, M., Malissen,B. lmmunol.4:529-91 1983. Genes of the major histo11. Yague,J., White, J., Coleclough,C., compatibility complexof the mouse. Kappler, J., Palmer, E., Marrack,P. Ann. Rev. Immunol.1:529-68 1985.TheT cell receptor: the alpha and 3. Flavell, R. A., Allen, H., Buddy,L. beta chainsdefine idiotype, andantigen and MHC specificity. Cell 42:81-87 C., Sherman, D. H., Waneck,G. L., Widera,G. 1986. Molecularbiology of 12. Ohashi, P. S., Mak,T. W., Van den the H-2 complex.Science 233:437-43 Elsen, P., Yanagi, Y., Yoshikai, Y., 4. Mellor, A. L., Weiss, E. H., RamaCaiman,A. F., Terhorst, C., Stobo, J. chandran, K., Flavell, R. A. 1983. A D., Weiss, A. 1985. Reconstitution of potential donor gene for the bmlgene an active surface T3/T-cell antigen conversion event in the C57BL mouse. receptor by DNA transfer. Nature316: Nature 306:792-95 606-9 13. Dembic, Z., Haas, W., Weiss, S., 5. Nathenson, S. G., Geliebter, J., Pfaffenbach, G. M., Zeff, R. A. 1986. McCubrey,J., Kiefer, H., von Boeho Murinemajor histocompatibility commer, H., Steinmetz, M. 1986. Transplex class-I mutants:molecularanalysis fer of specificity by murinealpha and and structure-function implications. beta T-cell receptor genes. Nature Ann. Rev. lmmunol.4:471-502 320:232-38 14. Adkins,B., Mueller,C., Okada,C. Y., 6. Zinkernagel, R. M., Doherty, P. C. 1974. Restriction of in vitro T-cell Reichert, R. A., Weissman, I. L., Spanmediated cytotoxicity in lymphocyte grade, G. J. 1987. Early events in Tchoriomeningitiswithin a syngeneicor cell maturation. Ann.Rev. lmmunol.5: semi-allogeneic system. Nature 248: 325-65 701-2 ¯ 15. Fink, P. J., Bevan, M. J. 1978. H-2 antigens of the thymusdeterminelym7. Zinkernagel, R. M., Doherty, P. C. 1979.MHC-restricted cytotoxic T cells: phocytespecificity. J. Exp. Med.148: studies on the biological role of poly766-75 morphicmajor transplantation anti16. Sprent, J. 1978. Roleof H-2geneproducts in the function of T helper ceils gens determining T-cell restrictionspecificity, function, and responfromnormaland chimericmice in vivo, siveness. Adv. Immunol.27:51-77 Immunol.Rev. 42:108-37 8. Dialynas,D. P., Wilde,D. B., Marrack, 17. Zinkernagel, R. M. 1978. Thymusand lymphohaematopoietic cells: their role P., Pierres, A., Wall, K. A., Havran, in T-cell maturationin selection of T W., Otten, G., Loken,M. R., Pierres, M., Kappler, J., Fitch, F. W.1983. cells’ H-2restriction-specificityandHCharacterization of the murine anti2 linked Ir genecontrol. Immunol.Rev. genie determinant, designated L3T4a, 42:224-70 recognized by monoclonal antibody 18. Kappler, J., Kubo, R., Haskins, K., GK1,5:expression of L3T4aby funcHannum,C., Marrack,P., Pigeon, M., tional T cell clonesappearsto correlate Mclntyre,B. Allison, J., Trowbridge, primarily with class II MHC antigenI. 1983. The majorhistocompatibility reactivity. Immunol.Rev. 74:29-56 complex-restrictedantigen receptor on T cells in mouseandman:identification 9. Swain, S. L. T cell subsets and the recognition of MHC class, lmmunol. of constantand variable peptides. Cell Rev. 74:129-42 35:295-302

Annual Reviews

Annu. Rev. Immunol. 1988.6:359-380. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

CLASSI ANTIGENSAND TUMOR GROWTH 19.Allison, J.P.,Ridge, L.,Lund, J., Gross-Pelose, J.,Lanier, L.L,Mclntyre, B.W.1984. Theroutine T cell antigen receptor andassociated structures. Immunol.Rev. 81:145-60 20. Saito, H., Kranz, D. M., Takagaki, Y., Hayday, A. C., Eisen, H. N., Tonegawa,S. 1984. A third rearranged and expressedgenein a clone of eytotoxic T lymphocytes.Nature 312: 3640 21. Brenner, M. B., McLean,J., Deno,D. P., Strominger, J. L., Smith, J. A., Owen,F. L., Seidman,J. G., Ip, S., Rosen,F., Krangel,M.S. 1986.Identification of a putative second T-cell receptor. Nature322:145-49 22. Bank,I., DePinho,R. A., Brenner,M. B., Cassimeris,J., Alt, F. W.,Chess,L. 1986.A functional T3moleculeassociated with a novel heterodimeron the surface of immature humanthymocytes. Nature322:179-81 23. Lanier, L. L., Federspiel, N. A., Ruitenberg,J. J., Phillips,J. H., Allison,J. P., Littman,D., Weiss,A. 1987. TheT cell antigen receptor complexexpressed on normal peripheral blood CD4-, CD8-T lymphocytes. A CD3-associated disulfide-linked y chain heterodimer. J. Exp. Med. 165:1076-94 24. Bluestone,J. A., Pardoll, D., Sharrow, S. O., Fowlkes, B. J. 1987. Characterization ofmurinethymocyteswith CD3-associatedT-cell receptor structures. Nature326:82-84 25. Chien, Y., Iwashima,M., Kaplan, K. B., Elliott, J. F., Davis,M.M.1987.A newT-cell receptor genelocated within the alpha locus andexpressedearly in T-cell differentiation. Nature327: 67782 26. Samuelson, L. E., Harford, J. B., Klausner,R. D. 1985.Identification of the componentsof the murine T cell antigen receptor complex.Cell 43: 22331 27. Weiss, A., Imboden,J., Hardy, K., Manger,B., Terhorst, C., Stobo, J. 1986.Therole of the T3/antigenreceptor complexin T-cell activation. Ann. Rev. Immunol.4:593-619 28. Matzinger, P. 1981. A one-receptor view of T-cell behaviour. Nature292: 497-501 29. Schrader, J. W.1982. A single T-cell receptor: a speculative review of the intrathymic generation and modulation of the repertoire. Thymus4:181207 30. Cohn, M. 1983. The T-cell receptor mediating restrictive recognition of antigen. Cell 33:657-69

375

31.Parham,P. 1984. A repulsive view of MHC-restriction. ltnmunol. Today5: 89-92 32. Goverman,J., Hunkapiller, T., Hood, L. 1986.Aspeculative viewof the multicomponentnature of T cell antigen recognition. Cell 45:475-84 33. Pernis, B., Axel, R. 1985. A oneand a half receptor modelfor MHC-restricted antigen recognition by T lymphocytes. Cell 41:13-16 34. Hunig,T. R., Bevan,M.J. 1982.Antigenrecognition by clonedcytotoxic Tlymphocytesfollows rules predicted by the altered-self hypothesis.J. Exp. Med. 155:111-25 35. Heber-Katz, E., Schwartz, R. H., Matis,L. A., Hannum, C., Fairwell, T., Appella, E., Hansburg,D. 1982. Contribution of antigen-presenting cell majorhistocompatibility complexgene productsto the specificity of antigeninducedT-cell activation. J. Exp.Med. 155:1086-99 36. Hedrick,S. M., Matis,L. A., Hecht,T. T., Samuelson,L. E., Longo, D. L., Heber,Katz, E., Schwartz,R. H. 1982. Thefine specificity of antigen andIa determinantrecognition by T cell hybridomaclones specific for cytochrome c. Cell 30:141-52 37. Kappler, J. W., Skidmore,B., White, J., Marrack, P. 1981. Antigeninducible,H-2-restrictedinterleukin-2producing T cell hybridomas.Lackof independent antigen and H-2 recognition. J. Exp. Med.153:1198-1214 38. Buus,S., Colon,S., Smith,C., Freed, J. H., Miles, C., Grey, H. M. 1986. Interaction between a "processed" ovalbuminpeptide and Ia molecules. Proc. Natl. Acad. Sci. USA83: 396871 39. Babbitt, B., Allen, P. M., Matsueda, G., Haber, E., Unanue, E. R. 1985. Bindingof immunogenic peptides to Ia histocompatibility molecules. Nature 317:359-61 40. Babbitt, B., Matsueda,G., Haber, E., Unanue,E. R., Allen, P. M.1986.Antigenic competitionat the level of peptide-Ia binding.Proc.Natl. Acad.Sci. USA83:4509-13 41. Buus,S., Sette, A., Colon,S. M., Miles, C., Grey, H. M. 1987. The relation betweenmajor histocompatibility complex (MHC)restriction and the capacity of Ia to bind immunogenic peptides. Science235:1353-58 42. Unanue,E. R., Allen, P. M.1987. The basis for the immunoregulatory role of macrophages and other accessorycells. Science 236:551-57

Annual Reviews

Annu. Rev. Immunol. 1988.6:359-380. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

376

TANAKAET AL

43. Gammon, G., Dunn, K., Shastri, N., Oki,A., Wilbur,S., Sercarz,E. E. 1986. Neonatal T-cell tolerance to minimal immunogenicpeptides is caused by donal inactivation. Nature319:413-15 44. Townsend,A. R. M., Rothbard, J., Gotch,F. M., Bahadur,G., Wraith,D., McMichael, A. J. 1986. Theepitopes of influenza nucleoprotein recognizedby cytotoxic T lymphocytescan be defined withshort synthetic peptides. Cell 44: 959-68 45. Parham,P., Clayberger, C., Zorn, S. L., Ludwig,D. S., Schoolnik, G. K., Krensky, A. M. 1987. Inhibition of alloreactive cytotoxic T lymphocytes by peptides from the a2 domain of HLA-A2.Nature 325:625-28 46. Prehn, R. T., Main, J. M. 1957. Immunity to methylcholanthreneinducedsarcomas.J. Natl. CancerInst. 18:769-78 47. Klein, G., Sjogren, H. O., Klein, E., Hellstrom, K. E. 1960. Demonstration of resistance against methylcholanthrene-inducedsarcomasin the primary autochthonous host. Cancer Res. 20:1561-72 48. Koeppen,H., Rowley,D. A., Schreiber, H. 1986. Tumor-specificantigens and immunological resistance to cancer. In Host-ResistanceMechanisms to Infectious Agents,Tumorsand Allografts, ed. R. M.Steinman,R. J. North, pp. 359-86. NewYork: Rockefeller Univ. Press 49. Doherty, P. C., Knowles, B. B., Wettstein, P. J. 1984. Immunological surveillance of tumorsin the context of major histocompatibility complex restriction of T cell function.Adv.Cancer Res. 42:1-65 50. Gooding,L. R. 1977. Specificities of killing by cytotoxic lymphocytesgenerated in vivoandin vitro to syngeneic SV40transformed cells. J. ImmunoL 118:920-27 51. Gooding,L. R. 1979. Specificities of killing by T lymphocytes generated against syngeneieSV40transformants: studies employingrecombinantwithin the H-2 complex. J. Immunol. 122: 1002-8 52. Knowles,B., Koncar,M., Pfizenmaier, K., Solter, D., Aden,D. P., Trinchieri, G. 1979. Geneticcontrol of the cytotoxic T cell response to SV40tumorassociatedspecific antigen. J. ImmunoL 122:1798-1806 53. Pan,S., Wettstein,P. J., Knowles, B. B. 1982. H-2Kbmutations limit the CTL response to SV40TASA.J. Immunol. 128:243-46

54. Gooding,L. R. 1982. Characterization of a progressive tumor from C3H fibroblasts transformedin vitro with SV40virus. Immunoresistance in vivo correlates with phenotypicloss of H2Kk. J. Immunol.129:1306-12 55. Kiessling, R., Wigzell, H. 1979. An analysis of the murineNKcell as to structure, functionandbiologicalrelevance, lmmunol.Rev. 44:165-208 56. Herberman, R. B., Ortaldo, J. R. 1981. Naturalkiller cells: their role in defence against disease. Science214:24-30 57. Karre, K. 1985. Role of target histocompatibilityantigens in regulation of natural killer activity: a re-evaluation and hypothesis. In Mechanisms of Cytotoxicity by NKCells, ed. R. B. Herberman,D. M. Callewaert, pp. 8192. NewYork: Academic 58. Karre, K., Ljunggren,H. G., Piontek, G. E., Kiessling, R. 1986. Selective rejection of H-2-deficient lymphoma variants suggest alternative immune defense strategy. Nature319:675-78 59. Stern, P., Gidlund, M., Orn, A., Wigzell,H. 1980.Natural killer cells mediate lysis of embryonalcarcinoma cells lacking MHC. Nature 285:341-42 60. Piontek, G. E., Taniguchi,K., Ljunggren, H. G., Gronberg,A., Kiessling, R., Klein, G., Karre, K. 1985. YAC-1 MHC class I variants reveal an association betweendecreasedNKsensitivity and increased H-2 expression after interferon treatment or in vivo passage. J. lmmunol. 135:4281-88 61. Raska, K., Gallimore, P. H. 1982. An inverserelation of the oncogenic potential of adenovirus-transformedcells andtheir sensitivity to killing by syngeneicnaturalkiller cells. Virology123: 8-18 62. Patek, P. Q., Lin, Y., Collins, J. L., Cohn,M.1986.In vivoor in vitro selection for resistance to natural cytotoxic cell lysis selects for variants with increased tumorigenicity. J. ImmunoL 136:741-45 63. Ljunggren,H. G., Karre, K. 1985. Host resistance directed selectively against H-2-deficient lymphomavariants. Analysis of the mechanism.J. Exp. Med. 162:1745-59 64. Ljunggren, H. G., Karre, K. 1986. Variations in MHC antigen expression on tumoursandits significance. Experimental strategies and interpretations in the analysis of changesin MHC gene expression during tumourprogression. Opposing influencesof T cell and natural killer mediatedresistance. J. Immunogenet. 13:141-51

Annual Reviews

Annu. Rev. Immunol. 1988.6:359-380. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

CLASSI ANTIGENSAND TUMOR GROWTH 65. Schmidt, W., Festenstein, H. 1982. Resistance to cell-mediated cytotoxicity is correlatedwith reductionof H-2Kgene products in AKRleukemia. Immunogenetics 16:257~4 66. Hailoran, P. F., Wadgymar, A., Autenfled, P. 1986. The regulation of expressionof majorhistocompatibility complexproducts. Transplantation41: 413-20 67. Ozato, K., Wan,Y.-J., Orrison, B. M. 1985. Mousemajor histocompatibility class I geneexpressionbegins at midsomitestage andis induciblein earlierstage embryosby interferon. Proc. Natl. Acad. Sci. USA82:2427-31 68. Tanaka,K., Appella,E., Jay, G. 1983. Developmentalactivation of the H-2K geneis correlated with an increase in DNAmethylation. Cell 35:457-65 69. Christy, B., Scangos,G. 1986. Control of gene expression by DNAmethylation. In MolecularGenetics of Mammalian Cells, ed. G. M. Malacinski. London: Macmillan 70. Graziano, K. D., Edidin, M. 1971. Serological quantitation of histocompatibility-2antigensand the determination of H-2 in adult and fetal organs. In Proceedings of the Symposiumon Immunogeneticsof the H-2 System, pp. 251-56.Basel: Karger 71. Mayrhofer, G., Schon-Hegrad,M. A. 1983. Ia antigens in rat kidney, with special referenceto expressionin tubular epithelium.J. Exp. Med.157: 20972109 72. Halloran, P. H., Jephthah-Ochola,J., Urmson,J., Farkas, S. 1985. Systemic immunologic stimuli increase class I andII antigen expressionin mousekidney. J. Immunol.135:1053-60 73. Steiniger, B., Klempnauer,J., Wonigeit, K. 1985. Altered distribution of class I and class II MHC antigens during acute pancreasallograft rejection in the rat. Transplantation 40: 23439 74. Lindahl,P., Leary,P., Gresser,I. 1973. Enhancementby interferon of the expression of surface antigens on murine leukemia L 1210 cells. Proc. Natl. Acad. Sci. USA70:2785-88 75. Wallach, D., Fellous, M., Revel, M. 1982.Preferentialeffect of y-interferon on the synthesis of HLAantigens and their mRNAs in humanceils. Nature 299:833-36 76. Friedman,R. L., Manly,S. P., McMahon, M., Kerr, I. M., Stark, G. R. 1984. Transcriptional and post-transcriptional regulation of interferon inducedgeneexpressionin humancells.

377

Cell 38:745-55 77. Yoshie, O., Schmidt,H., Lengyel,P., Reddy,E. S. P., Morgan,W.R., Weissman,S. M.1984. Transcripts of human HLAgenefragmentslacking the 5’ terminalregionin transfectedmousecells. Proc. Natl. Acad. Sci. USA81:649-53 78. Friedman,R. L., Stark, G. R. 1985. or-interferon induced transcription of HLAand metaalthionein genes containing homologousupstream sequences. Nature 314:637-39 79. Israel, A., Kimura,A., Fournier, A., Fellous, M., Kourilsky,P. 1986. Interferon response sequence potentiates activity of an enhancerin the promoter region of a mouseH-2 gene. Nature 322:743-46 80. Korber,B., Hood,L., Stroynowski,I. 1987. Regulation of murine class I genes by interferons is controlled by regions located both 5’ and 3’ to the transcriptioninitiation site. Proc.Natl. Acad. Sci. USA84:3380-84 81. Yarden,A., Shure-Gottlieb, H., Chebath, J., Revel, M., Kimchi,A. 1984. Autogenousproduction of interferon beta switches on HLAgenes during differentiation of histiocytic lymphoma U937cells. EMBO J. 3:969-73 82. Sanderson,A. R., Beverley, P. C. L. 1983.Interferon, fl2-microglobulinand immunoselection in the pathway to malignancy: a blinkered view from Nag’s HeadYard. Immunol.Today 4: 211-13 83. Collins, T., Lapierre,L. A., Fiers, W., Strominger,J. L., Pober, J. S. 1986. Recombinant humantumor necrosis factor increases mRNA levels and surface expression of HLA-A,B antigens in vascularendothelialcells anddermal fibroblasts in vitro. Proc.Natl. Acad. Sci. USA83:446-50 84. May,L. T., Helfgott, D. C., Sehgal, P. B. 1986.Anti-/~-interferon antibodies inhibit the increased expression of HLA-B7 mRNA in tumor necrosis factor-treated humanfibroblasts: structural studies of the f12 interferon involved. Proc. Natl. Acad. Sci. USA 83:8957-61 85. Ball, E. D., Guyre,P. M., Glynn,J. M., Rigby, W.F. C., Fanger, M. W.1984. Modulationof class I HLAantigens on HL-60promyelocytic leukemia cells by serum-free medium:re-induction by gamma-IFNand 1,25-dihydroxyvitamin D3(calcitriol). J. Immunol. 132:2424-28 86. Suthedand,J., Mannoni,P., Rosa, F., Huyat, D., Turner, A. R, Fellous, M. 1985. Induction of expression of HLA

Annual Reviews

Annu. Rev. Immunol. 1988.6:359-380. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

378

TANAKAET AL

class I antigenson K562by interferons and sodium butyrate. Hum.lmmunol. 12:65-73 87. Gorelik, E., Peppoloni, S., Overton, R., Herberman,R. B. 1985. Increase in H-2 antigen expression and immunogenicity of B16 melanoma cells treated with N-methyl-N’nitronitrosoguanidine. Cancer Res. 45:5341-47 88. Parmiani,G., Carbone,G., Invernizzi, G., Pierotti, M. A., Sensi, M. L., Rogers,M.J., Appella,E. 1979.Alien histocompatibility antigens on tumor cells. Immuno#enetics 9:1-23 89. Festenstein, H., Schmidt, W. 1981. Variation in MHC antigenic profiles of tumorcells and its biological effects. Immunol.Rev. 60:85-127 90. Festenstein, H., Garrido, F. 1986. MHC antigens and malignancy. Nature 322:502-3 91. Garrido, F., Schirrmacher,V., Festenstein, H.1976.H-2-1ikespecificities of foreign haplotypes appearing on a mousesarcoma after vaccinia virus infection. Nature259:228-30 92. Schmidt, W., Festenstein, H. 1980. Serological and immunochemical studies of H-2allospecificities on K36.16 syngeneic tumor of AKR.J. Immuno#enet. 7:7-17 93. Festenstein, H., Hui, K. M. 1986. AberrantH-2-1ikeallospecificities on K36.16 thymoma.Studies by radiobinding and immunoprecipitationwith anti-H-2 monoclonalantibodies. J. Immunoyenet.13:113-16 94. Hui, K. M., Minamide,L., Prandoni, N., Festenstein, H., Grosveld, F. G. 1986.Structural variations in the H-2 genes of AKRlymphomas. J. Immuno~enet. 13:117-21 95. Wortzel,R. D., Philipps, C., Schreiber, H. 1983.Multiple tumor-specificantigens expressedon a single tumorcell. Nature 304:165q57 96. Philipps, C., McMillan,M., Flood, P. M., Murphy, D. B., Forman, J., Lancki, D., Womack,J. E., Goodenow,R.S., Schreiber, H. 1985. Identification of a unique tumor-specific antigen as a novelclass I majorhistocompatibility molecule. Proc. l~atL Acad. Sci. USA82:5140-44 97. McMillan, M., Lewis, K. D., Rovner, D. M. 1985. Molecular characterization of novel H-2class I molecules expressed by a C3HUV-induced fibrosarcoma. Proc. Natl. Acad. Sci. USA 82:5485-89 98. Stauss, H. J., Linsk, R., Fischer, A., Watts, S., Banasiak,D., Haberman, A.,

Clark, I., Forman,J., McMillan,M., Schreiber, H., Goodenow, R. S. 1986. Isolation of the MHC genes encoding the tumor-specific class I antigens expressedon a murinefibrosarcoma.J. Immuno#enet.13:101-11 99. Linsk, R., Vogel, J., Stauss, H., Forman, J., Goodenow,R. S. 1986. Structure and function of three novel MHC class I antigens derived from a C3Hultraviolet-induced fibrosarcoma. J. Exp. Med.164:794-813 100. Gipson,T. G., Imamura,M., Conliffe, M. A., Martin, W. J. 1978. Lung tumor-associated derepressed alloantigen codedfor by the K region of the H-2majorhistocompatibility complex. J. Exp. Med.147:1363-73 101. Callahan, G. N., Pardi, D., Giedlin, M.A., Allison, J. P., Morizot, D. M., Martin, W.J. 1983. Biochemicalevidencefor expression of a semi-allogeneic, H-2antigen by a murineadenocarcinoma. J. Immunol. 130: 47174 102. Lilly, F. 1968. The effect of histocompatibility-2type on responseto the Friend leukemiavirus in mice. J. Exp. Med. 127:465-73 103. Chesebro,B., Wehrly,K., Stimpfling, J. 1974. Host genetic control of recovery from Friend leukemia virusinduced Splenomegaly. Mapping of a gene within the major histocompatibility complex.J. Exp. Med.140: 1457-67 104. Chesebro,B., Wehrly,K. 1976.Studies on the role of the host immune response in recoveryfromFriend virus leukemia. II. Cell-mediated immunity. J. Exp. Med. 143:85-99 105. Blank, K. J., Lilly, F. 1977. Evidence for an H-2/viral protein complexon the cell surface as the basis for the H-2 restriction of cytotoxicity. Nature269: 808-9 106. Schmidt,W.,Atfield, G., Festenstein, k geneproduct(s) H. 1979. Loss of H-2K from an AKRspontaneous leukemia. Immuno#enetics8:311-21 107. Schmidt, W., Alonso, A., Leben, L., Festenstein,H. 1981.Differentialregulation of H-2 antigen expression in tumorsandits biological consequences. Transplant. Proc. 13:1814-18 108. Schmidt, W., Festenstein, H. 1982. Resistance to cell-mediated cytotoxicity is correlated withreductionof H-2Kgene products in AKRleukemia. Irnmunogenetics16:2.57-64 109. Hui, K., Grosveld,F., Festenstein, H. 1984. Rejection of transplantable AKR leukemia cells following MHC DNA-

Annual Reviews

Annu. Rev. Immunol. 1988.6:359-380. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

CLASS I ANTIGENS AND TUMORGROWTH mediatedcell transformation. Nature 31l: 750-52 110. Meruelo, D., Kornreich, R., Rossomando,A., Pampeno,C., Boral, A., Silver, J. L., Buxbaum, J., Weiss,E. H., Devlin,J. J., Mellor,A.L., Flavell, R. A., Pellicer, A. 1986. Lackof class I H-2antigens in cells transformed by radiation leukemiavirus is associated with methylationand rearrangementof H-2 DNA.Proc. Natl. Acad. Sci. USA 83:4504-8 111. Meruelo, D., Rossomando,A., Offer, M,, Buxbaum,J., Pellicer, A. 1983. Association of endogenousviral loci with genes encoding murine histocompatibility and lymphocytedifferentiation antigens. Proc. Natl. Acad. Sci. USA80:5032-36 112. Meruelo, D., Kornreich, R., Rossomando,A., Pampeno,C., Mellor, A. L., Weiss,E. H., Flavell, R. A., Pellicer, A. 1984. Murine leukemia virus sequences are encodedin the murine major histocompatibility complex. Proc. Natl. Acad. Sci. USA81:1804-8 113. Rogers, M., Gooding, L. R., Margulies, D. H., Evans, G. A. 1983. Analysisof a defect in the H-2genes of SV40transformed C3Hfibroblasts that do not express H-2Kk. J. lmmunol. 130:2418-22 114. Lewis,A. M.Jr., Cook,J. L. 1984.The interface betweenadenovirus-transformed cells and cellular immune responsein the challengedhost. Curr. Top. Microbiol. lmmunol.110:1-22 115. Schrier, P. I., Bernards,R., Vaessen,R. T. M. J., Houweling,A., van der Eb, A. J. 1983.Expressionof class I major histocompatibility antigens switched off by highly oncogenicadenovirus12 in transformedrat cells. Nature305: 771-75 116. Vaessen, R. T. M.J., Houweling,A., Israel, A., Kourilsky,P., vantier Eb, A. J. 1986. AdenovirusE1A-mediated regulation of class I MHC expression. EMBOJ. 5:335-41 117. Tanaka,K., Isselbacher, K. J., Khoury, G., Jay, G. 1984. Reversal of oncogenesis by the expression of a major histocompatibility complexclass I gene. Science 228:26-30 118. Tanaka,K., Hayashi,H., Hamada,C., Khoury,G., Jay, G. 1986. Expression of major histocompatibility complex class I antigens as a strategy for the potentiation of immune recognition of tumorcells. Proc.Natl. Acad.Sci. USA 83:8723--27 119. Hayashi, H., Tanaka, K., Jay, F., Khoury,G., Jay, G. 1985, Modulation

379

of the tumorigenicityof humanadenovirus-12-transformedcells by interferon. Cell 43:263~7 120. Eager, K. B., Williams,J., Breiding, D., Pan, S., Knowles,B., Appella,E., Ricciardi, R. P. 1985. Expressionof histocompatibility antigens H-2K,-D and -L is reduced in adenovirus-12transformedmousecells andis restored by interferon V. Proc.Natl. Acad.Sci. USA 82:5525-29 121. Vaessen, R. T. M. J., Houweling,A., van der Eb, A. J. 1987. Post-transcriptional control of class I MHC mRNA expression in adenovirus 12transformedcells. Science 235: 148688 122. Sawada,Y., Fohring,B., Shenk,T. E., Raska, K. 1985. Tumorigenicity of adenovirus-transformedcells: region E1Aof adenovirus 12 confers resistance to natural killer cells. Virology 147:413-21 123. Mellow, G. H., Fohring, B., Dougherty, J., Gallimore,P. H., Raska, K. 1984. Tumorigenicityof adenovirustransformedrat cells and expression of class I majorhistocompatibility antigen. Virology 134:460-65 124. Hecht, T. T., Summers,D. F. 1972. Effect of vesicular stomatitis virus infection on the histocompatibility antigenof L cells. J. Virol. 10:578-85 125. Jennings, S. R., Rice, P. L., Klosazewski, E. D., Anderson, R. W., Thompson, D. L., Tevethia, S. S. 1985. Effect of Herpessimplexvirus type 1 and 2 surface expression of class I majorhistocompatibilitycomplexantigenson infectedcells. J. Virol. 56: 75766 126. Paabo,S., Nilsson,T., Peterson,P. A. 1986. Adenoviruses of subgenera B, C, D, and E modulate ceil-surface expressionof majorhistocompatibility complexclass 1 antigens. Proc. Natl. Acad. Sci. USA83:966.%69 127. Andersson,M., Paabo,S., Nilsson, T., Peterson, P. A. 1985. Impairedintracellular transport of class I MHC antigens as a possible meansfor adenoviruses to evadeimmune surveillance. Cell 43:215-22 128. Burgert, H. G., Kvist, S. 1985. An adenovirustype 2 glycoprotein blocks cell surface expressionof humanhistocompatibilityclass I antigens. Cell 41: 987-97 ¯ 129. Wallich, R., Bulbue, N., Hammerling, G. J., Katzav,S., Segal, S., Feldman, M. 1985. Abrogation of metastatic properties of tumourcells by de novo expression of H-2Kantigens following

Annual Reviews

Annu. Rev. Immunol. 1988.6:359-380. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

380

TANAKAET AL

H-2gene transf~tion. Nature315: 3015 130. Doyle, A., Martin, W. J., Funa, K., Gazdar,A., Carney,D., Martin,S. E., Linnoila,I., Cuttitta, F., Mulshine,J., Bunn, P., Minna, J. 1985. Markedly decreasedexpressionof class I histocompatibility antigens, protein, and mRNAin human small cell lung cancer. J. Exp. Med.161:1135-51 131. Holden,C. A., Sanderson,A. R., MacDonald, D. M. 1983. Absence of humanleukocyte antigen moleculesin skin tumors and some cutaneous appendages: evidence using monoclonal antibodies. J. Am.Acad.Dermatol. 9:867-71 132. Turbitt, M. L., Mackie, R. M. 1981. Lossof fl2-mieroglobulinfromthe cell surface of cutaneous malignant and premalignantlesions. Br. J. Dermatol. 104:507-13 133. Holden, C. A., Shaw, M., McKee,P. H., Sanderson, A. R., MacDonald,D. M.1984. Lossof fl2-mieroglobulinin eccrine porocarcinoma. Arch. Dermatol. 120:732-35 134. Lampson, L. A., Fisher, C. A., Whelan, J. P. 1983. Stalking paucity of HLA-A, B, C and fl2-mieroglobulin on human neuroblastoma cell lines. J. Imrnunol. 130:2471-78 135. Fleming, K. A., McMichael, A., Morton, J. A., Woods,J., McGee,J. O. D. 1981. Distribution of HLAclass I antigens in normalhumantissue and in mammary cancer.J. Clin. Pathol. 34: 779-84 136. Natali, P. G., Giacomini,P., Bigotti, A., Lami, K., Nicotra, M. R., Ng, A. K., Ferrone, S. 1983. Heterogeneityin

the expression of HLAand tumorassociated antigens by surgically removedand cultured breast carcinomacells. CancerRes. 43:660-68 137. Travers,P. J., Arklie, J. L., Trowsdale, J., Patillo, R. A., Bodmer,W.F. 1980. Lack of expression of HLA-A,B, C antigens in chorioearcinomaand other humantumor cell lines. Natl. Cancer 1nat. Monoyr.60:175-80 138. Umplyby, H. C., Heinemann, D., Symes,M. O., Williamson, R. C. N. 1985. Expressionof histocompatibility antigens and characterization of mononuclear cell infiltrate in normaland neoplastic colorectal of humans. J. Natl. Cancer Inst.tissues 74:1161~8 139. Nanni, P., Colombo,M., DeGiovanni, C., Lollini, P., Nicoletti, G., Parmiani, G., Prodi, G. 1983. Impaired H-2 expression in BL6melanomavariants. J. Immunogenet.10:361-70 140. Jones, E. A., Bodmer,W.F. 1980. Lack of expression of HLAantigens on choriocarcinomacell lines. Tissue Antigens 16:195-202 141. Brodeur,G. M., Seeger, R. C., Schwab, M., Varmus,H. E., Bishop,J. M. 1984. Amplification of N-mycin untreated neuroblastoma correlates with advanced disease stage. Science 224: 1121-24 142. Little, C. D., Nau, M.M., Carney, D. N., Gazdar,A. F., Minna,J. D. 1983. Amplificationand expressionof the cmyc oncogene in humanlung cancer cell lines. Nature306:194-96 143. Bernards, R., Dessain, S. K., Weinberg, R. A. 1986. N-mycamplification causes down-modulationof MHC class I antigen expression in neuroblastoma.Cell 47:667-74

Annu. Rev. Immunol. 1988.6:359-380. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Annu. Rev. Immunol. 1988.6:359-380. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Annual Reviews www.annualreviews.org/aronline

Annu. Rev. Immunol. 1988.6:381-405. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Ann. Rev. Immunol.1988. 6: 381-405 Copyright©1988by AnnualReviewsInc. All rights reserved

THE IMMUNOGLOBULIN SUPERFAMILY--DOMAINS FOR CELL SURFACE RECOGNITION1,2 Alan F. Williams and A. Neil Barclay MRC Cellular Immunology Unit, Sir William DunnSchool of Pathology, University of Oxford, Oxford OX13RE, United Kingdom ENCOUNTERING

THE Ig

SUPERFAMILY

WhenIg chains were first sequenced,segmentswithin the constant regions of Hand L chains showedsequencesimilarities, and this led to the idea that the Ig chains hadall evolvedfroma primordialgenecodingfor about 100 aminoacids (1). The domainswithin the Ig chains all contained characteristic intrachain disulfide bond,and the idea of the domainas an independentstructural unit was proposed(2). Thedomainhypothesis was firmly established whenthe structures of Vand C domainsweredetermined to reveal a common fold forming a sandwichof two fl-sheets that was stabilized by the conserveddisulfide bond(3, 4). Beta-2 microglobulin(fl2-m), identified in the urine of patients with kidneydisease, wasthe first nonimmunoglobulin structure found to share sequencesimilarities with Ig-domains.Thefl2-m sequencelooked like an Ig C-domain(5, 6). fl2-m wassubsequentlydiscovered to be part of the major histocompatibility complex(MHC) class I structure, and sequencing of the class I heavychain showedthat a segmentof sequenceadjacent to the transmembrane region was also similar to Ig C-domains(7, 7a). MHC antigens were knownto play somerole in the specificity of T lymphocyte t Abbreviations: NBRFdata base is a protein sequence data base from Protein Identification Resource (1987) National Biomedical Research Foundation, Washington, DC. Terms are defined in Table 1. z Because of limited space, full referencing for this article has not been possible. Recent key references that lead to the full literature on each topic are cited.

381 0732~)582/88/0410-0381 $02.00

Annual Reviews

Annu. Rev. Immunol. 1988.6:381-405. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

382

WILLIAMS & BARCLAY

recognition, and thus the finding that the MHCclass I was Ig-related was in accord with the concept that Ig domains were uniquely concerned with immune recognition. This view was deafly dominant in the 1970s. The Thy-1 differentiation antigen was sequenced at the same time as MHC class I H chain, and the finding that this molecule was like a single Ig V domainwas not consistent with the immunerecognition concept (8). Thy-1 was expressed in large amounts in neural tissue and thymocytes in rodents, but expression in lymphoidcells was not conserved in all species. It thus seemedlikely that Ig-related structures wouldhave a general role in cell surface recognition (9). In the 1980s, manynew cell surface structures have been identified and sequenced, and the Ig-related family of molecules can nowbe argued to include the structures shown in Figure 1 and Table 1. The molecules have a diversity of functions (Table 2), but in most cases the common denominatoris a recognition role at the cell surface. The genetic loci are widely spread throughout the chromosomes, but a number of interesting linkage groups are seen. This family of molecules is undoubtedly one of the key groups not only in immunity but also in the mediation of cell surface recognition to control the behavior of ceils in various tissues.

Fi~lure 1 Models for molecules in the Ig-superfamily. One model is shownfor each main molecular type from one species (Table 1), and in somecases the same modelsuffices for the additional structures namedin brackets. The circles showsequence segments that fold as for an Ig domainor are predicted to do so at least to the extent of two sheets with fl-strands ABE:GFC (Figure 2). Segments labelled V, C1, and C2 are in the categories indicated Figure 2 and in the text. Domainnumbers as used in the text and figures are from the NH 2terminus. In CD4four domains are counted even though the second domain is not typically Ig-related. In the MHC class I H chain and related structures, the three obvious segments starting from the NH2-terminus are called 0~1, ~2, and % while in MHCclass II fl and e chains, the segments are ill, f12 and cq, c~2. CD1is shownindependently from class I to indicate that it has muchlower sequence identity to class I than do Qa and T1 (full CDla data by personal communicationfrom L. H. Martin, F. Calabi, and C. Milstein). Intrachain disulfide bonds that are like the conserved Ig disulfide bond are shownby ~ symbols within the circles, and cases where these are confirmed are given in Table 1. Other intrachain bonds also exist, but these are mostly not shown. Interchain bonds are indicated by SS between chains knownto be disulfide linked. CTLA4 could well exist in a dimer since a free sulphydryl is predicted in a membraneproximal position. N-linked carbohydrate sites as indicated by the presence of an Asn x Thr or Ser sequence are shown by the symbol ($) unless absence of glycosylation is known. The presence of glycophospholipid anchors is indicated by an arrow for Thy-l, LFA-3, and one NCAM form, and the possibility of this is indicated for CEAby an arrow plus ?. The Qa2 antigen also has a lipid tail (see text). A second form LFA3with a protein anchor has also been identified (84). References are in Table

Annu. Rev. Immunol. 1988.6:381-405. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Annual Reviews THE IMM-LINOGLOBULIN

IgM

CD1 TcR: CD 3 Complex

LFA3 CD2

TTT~TTTTr TTYT,LT-TTTTT<

SUPERFAMILY

MHCCLASSI (TL)

383

CLASS

MHC

Annual Reviews 384

W~LLIAMS & BARCLAY

T~ble1 Moleculesof the Ig supeffamily

Annu. Rev. Immunol. 1988.6:381-405. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Category

Ig-like Sequence Human disulfide number chromosome bonds References

Immunoglobulins H chains (IgM) 572 (M) 14q32.33 L chain kappa 214 (M) 2p12 L chain lambda 213 (M) 22ql1.12 T cell receptor (Tcr) complex Tcr ~-chain 250 (M) 14ql1.2 fl-ehain 282 (M) 7q35 y-chain 286 (M) 7pl 5 X-chain 272 (M) ND CD3y-chain 160 (H) 1 lq23 6-chain 150 (H) 1 Iq23 e-chain 185 (H) 1 lq23 Majorhistocompatibility complex (MHCantigens) Class I H-chain 339 (H) 6p21.3 fl2-m 99 (H) 15q21-q22 ClassII ~t 229 (H) 6p21.3 fl 237 (H) 6p21.3 /32-massociatedantigens TLH chain 335 (M) mouseonly Qa H chain 313 (M) mouse only CDla H chain 311 (H) 1 T cell adhesionmolecules lp13 CD2 322 (R) LFA-3 207 (H) 1 T subset antigens CD4 435 (H) 12pter.pl2 CD8chain I 210 (R) 2p12 chain II 187 (R) 2p12 CTLA4 188 (M) ND Brain/lymphoidantigens Thy-1 lll (R) 11q23 MRCOX-2 248 (R) Immunoglobulin receptors Poly Ig R 755 (RB) Fcy2b/),lR 351 (M) Neural molecules Neural adhesion molecule (NCAM) 1072 (CH) 11q23 Myelin associated gp (MAG) 610 (R) 219 (R) Po myelinprotein

Y Y Y

(2,10)

ND ND ND ND ND ND ND

(11)

Y Y Y Y

(7, 16)

ND ND ND

(18) (19, 20) (21)

ND ND

(22, 23, 24) (22, 25, 26)

Y ND ND ND

(27, 28) (27) (27, 29) (30)

Y ND

(8, 31, 32) (33)

Y Y

(34, 35, 36) (37, 38, 39)

ND ND ND

(40, 41) (42, 43) (42, 44)

(12) (13, 14, 15)

(6) (17)

Annual Reviews THE IMMUNOGLOBULIN SUPERFAMILY

385

Table 1 (continued)

Annu. Rev. Immunol. 1988.6:381-405. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Category Tumor antigen Carcinoembryonic antigen (CEA) Growth factor receptors Platelet-derived growth factor (PDGF)receptor Colony stimulating factor-1 (CSF1) receptor Non-cell surface molecules (g iB-glycoprotein Basement membranelink protein

Sequence Human number chromosome

668

Ig-like disulfide bonds References

(H)

ND (45)

5q31-q32

ND

(42, 46)

953 (H)

5q33.2-q33.3

ND

(42, 47)

474 (H) 339 (R)

ND ND

Y Y

(48) (49, 50)

1067 (M)

Mostsequencesare predicted fromcDNA, but all havebeenalso characterized as proteins exceptTcr Xchain and CTLA4. Thesequencelength is for the fully processedformof one sequencein each category, and most sequencedata is from the NBRF data base. For LFA-3and CEA,the residue numberincludes the hydrophobic COOH-terminal sequencethat will be processedoff if a lipid tail is present. Differential exonsplicingof genes,givingalternative products,is seenfor Igs (membrane and secretedforms),polyIgR (2 forms), class I MHC and related molecules, NCAM (3 forms), MAG (2 forms), and CD8chain forms).Theletters in brackets after the sequencenumberindicate speciesas follows:M,mouse;H,human; R, rat; RB, rabbit; CH,chicken. The humanchromosome assignments are from Ref. (51) or other referencesgiven in the table. Underthe headingl#-like disulfide bondsa Yindicatesthe presenceof conserveddisulfide bondsas expectedfromsequencesimilarities, and NDdenotes"not determined."

CHARACTERISTICS

OF THE Ig-FOLD

AND

SEQUENCE PATTERNS Thetwofl-sheets of the Ig-foldconsistof anti-parallelfl-strandscontaining 5-10 aminoacids. Betweenthe sheets a hydrophobic interior is formed fromin-pointinghydrophobic aminoacids that alternate in the//-strands without-pointinghydrophilicresidues(3, 4). Theinteractionbetweenthe sheetsis furtherstabilizedby the conserved disulfidebond.TheIg fold for V- andC-domains is shownin Figure2, andthe core of the fold consists of ]/-strands A, B, E in one sheet andG, F, C in the other (52). These strandscomefromthe first andlast parts of the domainsequence,while in the middleconsiderablevariation in sequencelength occurs. V and V-related domainshave about 65-75 aminoacid residues betweenthe conserved disulfidebond,andthereare four]/-strandsin each]/-sheet plus a short fl-strand segmentacross the top of the domain.In C-domains the sequencebetweenthe disulfide bondis shorterat 55-60 residues,yielding sheets with4 and3 ]/-strands. In somenon-Igdomains (see below)as few as 40 residuesexist betweenthe disulfide bond,andin somecases the fold

Annual Reviews 386

WILLIAMS & BARCLAY

Annu. Rev. Immunol. 1988.6:381-405. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Table 2 Functions of the Ig-related molecules (References in Table 1) Moleculesand tissue expression

Functions

Recognitionwithin the superfamily

Immunoglobulins: B lymphocytesonly

B lymphocyteantigen receptors and in secretedform,antibodies

T cell receptors: T lymphocytesand thymocytes

T lymphocyteantigen receptors: no knownsoluble forms

CD3chains: ~tes and

Part of the Tcrcomplex;role in signal transduction?

No,antibodies recognizeantigen without involvementof other molecules Yes, heterophilic; Tcr binds MHC antigens plus peptide but recognitiondoes not involve Ig-related MHC segments CD3associates with Tcr but no knownrecognition of other molecules Yes, heterophilic;Tcrinteracts witt class I and class II MHC antigen

thymocytes

MHC antigens: Manycell types, inducedby interferon fl2-massociatedantigens: Subsetsof lymphoidceils T lymphocyteadhesion ~lecules: ~ocytes and T cells (some maerophages in rat); LFA-3, widespreadexpression T subset markers: CD4and CD8or~ thymocytesand T subsets, CD4on macrophages, CD8on NKcells; CTLA4, activatedTcells Brain/lymphoidantigens: Thy-1,neurons,fibroblasts, various lymphoid; MRC OX-2, neurons, endothelium,various lymphoid Immunoglobulin receptors: PolylgR,gut and liver epithelium; Fc),2b/),l R, macrophages Neural-associatedmolecules: N--’CAM, neuronsand glia, early embryo; MAG,peripheral and central myelin, someneurons;P0 peripheral myelin CEA: Ep~elialcells and their tumors, early embryos Growthfactor receptors: PDGFR,widespread on mesenchymal cells; CSF1R, monocytelineage Linkprotein: Basement membrane ct iB-glycoprotein: Foundin serum

Present peptides fromforeign antigento the Tcr; somesoluble forms Functions not known

Nonatural ligands known

CD2 of T cells interacts with LFA-3on other cells in adhesion reactions, Anti-CD2antibodies cantrigger Tcell division

Yes, heterophilic; CD2binds LFA-3

CD4and CD8appear to control the bias of Tcells towardsinteraction with class I or class II MHC. CTLA4function unknown

Perhaps heterophilic: CD4and CD maybind class II and class I MH antigens respectively. CTLA4 unknown

Anti-Thy-1 antibodytriggers mouse T lymphocytedivision; MRC OX-2function unknown

Nonatural ligands known

PolylgRtransports multimericIgA or lgMacross epithelium; macrophage Fc~,2b/~lR binds aggregated IgG NCAM mediates adhesion of neural cells. MAG mayfunction in myelination.P0 constitutes 50% of peripheralmyelinprotein

Yes, heterophilic for bothPolylgr and Fc~,2b/~,lR. First domainof PolylgRbinds IgA

Tumormarker but function unknown

Natural ligand unknown

Interact with growthfactors to trigger cell divisionandother activities

No, PDGFRand CSFIR not knownto react with molecules other than growthfactors

Acts as a binding moleculebetween proteoglycanand hyaluronate chain Function unknown

No

Yes, homophilic for NCAM via Ig-related parts and perhaps for P0. MAG not known

Natural ligands unknown

Annual Reviews

Annu. Rev. Immunol. 1988.6:381-405. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

THE IMMUNOGLOBULIN SUPERFAMILY

387

mayconsist of 3/~-strands in each sheet, plus a short connecting sequence across the top of the/~-sheet sandwich. Interactions between domains in Igs occur between the faces of the sheets, while in the interaction between antibody and antigen the contact residues consist of sequences in bends at the end of the V-domains(53). Thus, it seems that Ig-domains can interact with other molecules via any accessible part of their surface. The Ig-related domains of non-Ig molecules are described as being Vor C-like according to whether they are likely tO have a pattern of strands approximating to a V- or C-domain. A designation of a sequence as being V-like does not indicate sequence variation in the molecule concerned. Conserved patterns of sequence are seen among the Ig-superfamily domains, and some alignments are shown in Figure 2. The first and last /%strands have been omitted to simplify the data, but this does not imply that conserved patterns are absent in these regions. Also, the wholeof the domainis included in statistical analyses. The sequences are groupedinto three categories called the V-SET, C1-SET, and C2-SET(54). The V-SET includes antigen receptor V-domainsand other sequences likely to have a V-type fold. The extra sequences that form or are thought to form the C’ and C"/~-strands are obvious in Figure 2. The C1-SETincludes mostly receptor C-domains and MHCantigen domains, and these are distinguished from the C2-SET in some conserved sequence patterns. However, both C1-SETand C2-SETsequences are likely to be folded as for Ig C-domains. Across all these sequences, identities or conservative amino acid substitutions are seen in ]Lstrands B, C, E, and F; in particular, the alternating hydrophobic residues are evident. In regions outside the/%strands, conserved patterns characterize V-SETand C1-SETsequences (marked in Figure 2). The C2-SETsequences seem somehowin between the V-SET and C|-SETsince they are likely to have a C-type fold, but their sequence patterns in the region of ]~-strands E and F are like those of the V-SET. In Figure 1 all the domains are labelled as V, CI~ or C2, and these assignments are mostly clear-cut. However,in some cases the designation is somewhat arbitrary, and among the C-like domains, sequences are placed in the C2-SETunless they clearly show the conserved C1-SET residues. The one exception to this is the C domain of TCRalpha chain which is not typical of sequences in the C1 or C2-SET. In this case, assignment to the C1-SETwas made on the basis that the alpha chain is part of the heterodimer that dictates antigen specificity in one category of Tcrs. In general, statistical analysis of sequence similarities (see below) supports the domain assignments shownin Figure 1.

Annual Reviews 388

WILLIAMS & BARCLAY

~D%rurVbetarU~valphalg[gvlam~av (l)hea~ FT ~RSLTsB ASi

~cox-21~l

PISG-V~GSTFSNDH[]~K_DsA-TGAV-TTSNYAN~VQ~-~KP-DHLFTO~IOGTNNRAPGVP--KSB~Y~WoH______I YYT~E~MIKES||~_LF~YR~jTMM__RGLE~}~}~JVy PP--GRGLEWIIIS_OKAPKA~MSIFSNOEK___I --KILGE~GE~LI~FNNNVPIDDSG ~V LTK-F YHGoPT SEKD DT

g~ 11111

Annu. Rev. Immunol. 1988.6:381-405. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

IVT~0EEEAV--O~E

~ETT~

- b S

PDGFR(III) ~BA(IV) ~EA(V}

I

~ P -

L~T

I ~ - G V V EIQBTT-

P~

~ Q

R

D

G

V~

......................

N F Q~T Y P R M K - - - S G R L V Y~W~VNN~SLP ................... QyS~LIDG

LS

~D~e~llon

M

T~SEA

~ S -

E I

L~Q

H N D

K N I G G

D ................

¯ - V~T~V~A~]K A D S S P heavy (IZI)

-~]T -

~L ¢L~cl&L~JKiO iPLY] el

S

N S G A L

T ....................

A E E S~Q P .................... - VLy~E ~SlWlW v ~1_~1~1 E ................... -lIl

-I~]~

~L~J

"~E

D L

K~E

.....................

~ ~ ~I~ D ~ ........ R G E Q E

-~W~M~M

Q

: : ~ : .......

............

C r-~l~

~ I)IS~JI-~DE

I~OI~D~

(BOXED

RESIDUES

MATCH BOXES IN V-, C1- OR C2-S]

CD2 (I) PDGFR (IV) CEA (1)

vvF EA V H N - -

~

MP .............. --S~LIWILKDSRTLGDS a Q H L F G Y S~Y K G E R V D G N- R~ ~ I G~V I G T ~ ~ AT p G

V domain

v

", ’,,~---S-S,-~ ," ,’ C A B C D E" F" G~" I~-strandsalongV andC domains

F~c~B

C domain

~ C

I-sh~et~Folding patternfor V andC domains (d)

Annual Reviews

389

THE IMMUNOGLOBULINSUPERFAMILY ¯ POSITIONS CNA~CTERISTIC OF ~ V-SET SEQUENCES D

E

NQ SS-V AA T V V

VT, D S ........... FTIHLNKA ...........

EEG PED

PSAKMP

F E&EAI

F

L

SLN R RD-S PS L L MTL V F SSF NASF QP-S PR DT I EVED Q O N~PILIIII!K~- L K I E LPP KIMD-VKPE F F MVO IKH-L LN SGS ~TAK E TV Q V I F W - T L L N T L DIDIEIGIC~MICIL F N M W DG IV H -LDYS TFT DVKN PI A D~M ~ LRV ~

............

~

KG ITLQI ST ......... ~K D~I N I T E G ............ F K EI~I ~WVODP S ........... ~RS V LPSDR .............

I~ V lambda Ig V heavy Tcr V alpha Tcr V beta CD4 (I) ~RC OX-2 (I)

POSITIONS CONSERVEDACROSS THE SUPERFAMILY ~DQ S~T~K EIQE PE EA -~PA DIALEYT~A~E ~V ~T~S ~ NCAM (IV}

Annu. Rev. Immunol. 1988.6:381-405. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

L D_- - - M V L R E A V S~lV V R S R_Q I~AT V I Y ............. ARV SAE SL$ ~L P V T D Y~F

G

E ............ ............ ~ ~ , , , , , -- -- T T N V S V V P S ............ R I G S I[~[HI P T ~ S~ [LJ~

MA6(IV) PDGFR (IZI) CEA(V) Alpha1 B-OP FC.72b/71R CD3 eps~1on CD2(II)

S G N S

S V T E D S K .....

S

S

T

L S K- A D

K V

E

T

T I L D S Q E G D T~K T ..... ~M ~WIL~-~-E R ~ M G K HE H~ [~ 1~ ]Q ~I E K H S D L S F ........ S KD W S FY L Y YT E - F T P EK D q

G U I L P S A ..... ~W

Y~R

A

T~E~A

A

-

-

-

G

K

A

A

D

L~R~K~

K

Ig C laNbda Ig c kappa Ig C heav7 (] Ig c heavy (] Tcr C beta Tcr C gamma

CD1 alpha

Figure2 Alignments of Ig-related sequences and Ig-folding patterns. Sequences are aligned by eye and with the use of the ALIGNprogram (55), and sequence categories are defined the text. The positions of the fl-strands knownfor Ig V and C domains are indicated above and below the V-SETand C1-SET sequences, respectively. Sequences in regions corresponding to fl-strands A and G have been omitted to simplify the data. Someconserved sequence positions are indicated by symbols, and these often involve conservation of amino acids of similar type rather than identities. The sequences are referenced by NBRFprotein data base code in square brackets or literature references in round brackets. Ig V lambda, Mouse [L1MS4E]; Ig VH, human [G1HUNM]; Tcr V alpha, mouse [RWMSAV];Ter V beta, human [RWHUVY];CD4, human [RWHUTA];CD8 chain II, rat (56); PolyIgR, rabbit [QRRBG]; MRCOX-2, rat [TDRTOX];P0, rat (44); Thy-1, rat [TDRT]; NCAM, chicken (40); MAG,rat (42); PDGFR,mouse (46); CEA,human (45); AlphalV-gp, [OMHU 1B]; Fc72b/71 R, mouse (37); CD3epsilon, human(14); CD2, rat (24); Ig C lambda, human [L2HU]; Ig C kappa, human [K3HU]; Ig C heavy, human [GHHU]; TcR C beta, human [RWHUCY]; TcR C gamma, mouse [RWMSC1]; flz-M, human [MGHUB2]; MHC I alpha 3, human [HLHUB2]; MHCII beta 2, human [HLHU3D]; CDla alpha 3, human (21); LFA-3, human(25). Belowthe sequence alignments diagrams of the folding patterns of V and C domains are shown. The folds as determined for an Ig V~ and Ce domain are from Ref. (3), and the labelling of the fl-strands along the domain is illustrated in the schematic diagram. The schematic view of the fold at bottom right is adapted from Ref. (4).

3

Annual Reviews

Annu. Rev. Immunol. 1988.6:381-405. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

390

WILLIAMS & BARCLAY

Althoughconservedsequencepatterns are seen in Figure 2, no residue is invariant in all Ig-related domains.Theconserveddisulfide bondwas once considered the hallmark of Ig domains,but recently a functional antibody has been described that has a Tyr residue instead of Cys in rstrand F of the VHdomain(57). Also, it can be argued that domains CD2, LFA3, CD4, CEA, PDGFR,and CSF1Rmay be Ig-related even thoughthey haveno Cysresidues in putative fi-strands B and F (54, 58). Four of these sequencesare shownbelowthe C1-SET in Figure 2. In these and the other cases the Cysresidues are replaced by hydrophobicamino acids that wouldpresumablybe suitable as inpointing residues that stabilize an Ig-like fold, just as do the other hydrophobic residues in the rstrands. Thereis great diversity of sequencein the Ig-related molecules,and the question arises--why is a conservedpattern seen at all? Thebiological functions (see below)require uniquerecognition specificities, and these cannot directly be responsible for conservedsequencepatterns. Theconserved sequencesare mostlyseen in the r-strands that makeup the core of the fold. It canbe arguedboth that the fold itself is selectedfor, because it is stable to proteolysis,andthat this is an essential feature for molecules operating in the extracellular environment (54). CRITERIA FOR INCLUSION THE Ig SUPERFAMILY

OF MOLECULES IN

Theinitial argumentfor related domainswithin the Ig chains wasbased on sequencesimilarities. However,an evolutionary relationship between Vand C domainswasonly acceptedwhensimilarities in tertiary structures wereestablished. It remainsa consensusthat the first criterion for an Ig relationship should be the presence of a domain-sizedsequencewith significant similarity to Ig or Ig-related domains,but in addition there should also be the probability that the sequenceshares key structural featuresof the Ig-fold. If evolutionaryselection acts on the basis of domain stability, then a rationale for requiring sequenceandstructural similarity is evident. Toevaluatesequencesimilarities, a statistical test mustbe used, andthe ALIGN programof Dayhoff and colleagues (55) is nowwidely available as part of the NBRF data base package[Protein Identification Resource (1987) Protein SequenceDatabase, National BiomedicalResearch Foundation, Washington, DC]. The ALIGNprogram scores the best match betweentwo sequenceson the basis of a scoring matrix derived by determiningthe frequencyof aminoacid replacementsin equivalent molecules

Annual Reviews

Annu. Rev. Immunol. 1988.6:381-405. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

THE IMMUNOGLOBULIN SUPERFAMILY

391

betweenwidely divergent species. Thenthe sequences are scrambledand rescored a numberof times (e.g. 150) to yield a meanbest randomscore andstandard deviation (SD). Thescore for the real sequencesis expressed as the numberof SDunits awayfrom the randommeanscore. Assuming a normaldistribution and no effect of sequenceselection, scores of 3.1, 4.3, and5.2 SDunits indicate chanceprobabilities of 10- 3, 10-s, and-7, 10 respectively. In choosingsequencesfor ALIGN analyses, a putative domainis defined by taking sequencewithin positions that are 20 residues before and after Cysresidues that mightapproximateto the conservedIg disulfide bond. If Cysresidues are absent, then possible replacementsare identified and used to define the domainsegment.Theselection of Cysresidues to define the domaincarries the possible problemthat this will bias ALIGN scores since matches betweenCys residues carry a high value in the Dayhoff scoring matrix. Tocheck this effect, 11 segmentsfrommembrane molecules that are not Ig-related werechosenon the basis of the presenceof a suitable pair of Cysresidues. Thesewerescored against Ig-related domains(54). From682 scores, a meanand standard deviation of 0.6___1 SDunits was obtained. In the controls there werethree scores of > 4 (4.1, 4.2, 4.2) and 13 of > 3 < 4 SDunits. In testing a newsequence, ALIGN scores should be determinedwith as manydistinct sequences as possible from the V-SET,C1-SET,and C2SET. This overcomes the problem that the ALIGNprogram scores throughout the sequenceand takes no account of the conservedsequence patterns that havegreat significance whenassessing sequencesimilarities by eye (Figure 2). By chance a reasonable score might result in one comparison,but repeated goodscores should indicate that a test sequence contains a conservedIg-related pattern of sequence,since this is the only common denominatorbetweenthe sequencesin Figure 2 (54, 58). In Table 3 some ALIGNscores are shownfor fl2-m, Thy-1, NCAM, and LCA(control) against sequences from the V-SETand C1-SET. scores well with the sequencesfromthe C1-SET, but a significant relationship is not seen with V-SETsequences, and THY-1showsgood scores in the opposite direction. NCAM gives good scores with both C1-SETand V-SETsequencesbecause, although its length matcheswith C-domains,it has someof the conserved sequence patterns of the V-SET.The control sequence from LCAgives no good scores even though the sequence has two Cysresidues that can matchwith those in the Ig-domainsand it also has a Trp that can matchthe conservedTrp of t-strand C in Figure 2. Somemoleculesthat have been claimedto be Ig-related fail the ALIGN test against the sets of domains(54, 58). Theseinclude the adenoviral glycoprotein (59) and the CD5antigen (60). Also, the enzymesuperoxide

Annual Reviews

392

WILLIAMS & BARCLAY

Annu. Rev. Immunol. 1988.6:381-405. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Table 3 ALIGNscores for comparisons of Ig related sequences V or Vrelated

fl2-m

NCAM Thy-1 (III)

LCA

C or Crelated

fl2-m

NC.AM Thy-1 (III) LCA

Ig lambda Ig kappa Ig heavy Tcr beta Tcr alpha Tcr gamma CD8(chain I) CD4(I) PolylgR (I) PolylgR(III) MRCOX-2 (I) P0 Protein

-0.9 1.7 1.1 1.8 2.1 -0.2 2.6 2.4 1.5 1.7 - 1.1 0.7

7.4 3.7 3.9 3.3 2.3 1.6 4.5 2.5 5.7 5.8 5.0 3.5

-0.6 -0.1 1.5 -0.2 - 1.5 1.4 -0.3 -0.1 0 0.9 -0.6 -0.4

Ig lambda Ig kappa Ig CH1 Ig CH2 Ig CH3 Tcr beta Tcr alpha Tcr gamma MHCI 0e3 MHCII ct2 MHCI1 f12 CDI~t3

5.6 6.0 4.0 2.4 6.3 4,4 2,1 1.9 8.2 11,2 11.3 9. I

1.4 1.3 3.0 2.9 3.1 2.3 -0.3 0.8 2.2 3.7 2.4 1.3

3.3 5.4 3.9 4.6 4.4 3.9 4.7 5.5 2.7 4.3 5.3 6.0

4.7 4.0 4.1 3.8 3.7 3.0 1.7 3.6 2.9 4.9 4.3 5.4

- 1.£ - 1.5 0.7 0 0.~ 2.~ -0.~ -0.~ 1.." 15 1.~ IS

Domains weredefinedfroma position20 residuesbeforethe first Cysto 20 residuesafter the secondCysof a putati Ig-like disulfide bond.Theleucocytecommon antigen(LCA) sequenceis a controlandincludesresidues88-189fro the partial rat LCAsequence(54). All other sequencesare referencedin Figure2 exceptthe followingwith NBI~ data base code given in squarebracketsor referencein parentheses:Ig kappa[K1HURY], Tcr gamma V [RWMSV CD8chain I (56), Tcr alpha C [RWHUAC], MHC class I [HLHU12],MHC class II beta [HLHU3D]. The ALIG program (55) wasrunwith a bias of 6 and/~breakpenaltyof 6, and150random runswereperformed.

dismutasewhichhas a fold like an Ig-domain(61) showsno sequence similarity andthus is not regardedas beingin the Ig-superfamily (58). Otherdomains withno statistically significantrelationshipare the a~ and a2 domainsof MHC class I antigen and the ai and fl~ domainsof MHC class II antigens. A possible Ig-related segmentin the sequenceof HIV glycoproteingp 110 doesnot includesequencethat can be matchedwitha full domain(62). However,there are matcheswith Ig C-domains over residues that give ALIGN scores of 7-9 SD.This is sufficiently high to raise the possibility that the viral sequencehas beencapturedfromIgs withoutthe maintenance of the full domain.A sequencesimilarity might remainif it wereselected for unknown reasonsor if the captureof a piece of Ig-relatedsequenceby virus wasa recent event. Structural proof for an Ig-related domaincan only convincinglybe established by tertiary structure determinedby X-raycrystallography. Thusfar, this is only reportedfor Ig V andC domains andfor the fl2-m and a3 domainsof MHC class I antigens whichhave structures exactly like Ig CH3domains(6, 7a). Unambiguous but limited evidence comes fromthe determination of disulfide bonds.Thusfar in all cases wherethis has been determinedthe bondingpattern is in accordwith the Ig-fold (Table1). Circulardichroismcan convincinglyestablish the presence purefl-structurewithouta-helix, andthis has beenshownfor Thy-1(63).

Annual Reviews

THEIMMUNOGLOBULIN SUPERFAMILY393 Secondarystructure prediction can be used, but this remains imprecise (64) and in our view should be used as a test to see whether a domain assignment indicated by sequence similarities might be improbableon structural grounds. Finally, the exon pattern in the genes can support domainassignments,and this is discussed below.

Annu. Rev. Immunol. 1988.6:381-405. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

OVERALL

MOLECULAR

CHARACTERISTICS

TheIg superfamilyis notable becauseno extracellular sequenceincludes an enzymaticactivity or segmentsfrommorethan one protein superfamily. This generalization does not apply to intracellular parts since the PDGFR and CSF1Rmoleculeshavecytoplasmicdomainsthat havetyrosine kinase enzymaticactivities and are related in sequenceto other tyrosine kinase domains(46). Mixingof segmentsfrom different superfamilies is commonlyseen in other cell or immune-related surface molecules.For example, in the complement proteins, repeats of a disulfide-linked domainof about 60 aminoacids can be found together with serine protease domains,plus in somecases epidermalgrowthfactor-like segments(65). TheIg-related moleculescommonly form dimers(Figure 1), and binding betweendomainscan be homophilic, as seen betweenthe CH3domainsof Igs, or heterophilic, as in interactions betweenV domainsand Ig CH1and CLdomains.Stable dimersare often disulfide-linked, but this is not the case for the MHC antigens and fl2-m associated antigens. Metastable interactions can also occur, as is seen in the Tcr complexwherethe 0t and fl chains in the disulfide-linked dimerassociate with the CD3e, y, ~, and ¢ chains (13). (The~ chain is not shownin Figure1 since the sequence unknown.)This precedentraises the possibility that even weakerinteractions that cannot be detected by conventional techniques might occur between Ig-related molecules at the cell surface during functional responses. Carbohydratestructures can be dominantfeatures in someIg-superfamily molecules, and in CEAup to six possible N-linkedsites have been observedon one Ig-related domain(Figure 1). Betweentissues the same protein can be differentially glycosylated, and all the complexN-linked structures of Thy-1differ betweenbrain and thymus(66). Also on NCAM differences occur in glycosylationbetweenfetal and adult forms(40). The fetal formsshowextensive polysialation, and this is thought to modulate the adhesive potential of NCAM molecules. The transmembranesequences and cytoplasmic domainsof Ig-related moleculesshowgreat diversity. Mostmoleculeshave a hydrophobictransmembraneprotein sequence, but for Thy-1 (31), one form of NCAM (40, 41), and of LFA-3(25, 84) and Qa-2 antigen (20, 20a) membrane

Annual Reviews

Annu. Rev. Immunol. 1988.6:381-405. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

394

WILLIAMS & BARCLAY

attachment via a glycophospholipidanchor has been established. Such an attachment mayalso be the case for CEA,since this molecule has hydrophobicresidues at its COOH-terminus, but there are no basic residues (as is to be expected)on the cytoplasmicside of an authentic transmembranesequence (45). The COOH-terminalsequence of CEAis like that of Thy-1, whichis absent from the maturemoleculeand is presumed to be cleaved off whenthe glycophospholipid tail is attached (31, 67). similar sequence is also predicted from NCAM cDNAclones that are thought to encode the form of NCAM that has a glycophospholipidanchor (40, 41; reviewedin 85). Ig-superfamily molecules with predicted transmembranesequences showonly one such sequenceper chain, and in mostcases these sequences showno aminoacids with amideor chargedresidues. However,in all the CD3chains there is one acidic residue in the midst of the hydrophobic domain(13, 14), and in Tcr chains basic residues are found in similar positions (11). In Tcr ~ and X chains two basic residues and an Asnare foundin the 22 residues mostlikely to cross the bilayer (12). Thecharged residues in the hydrophobicdomainsof the Tcr complexmaystabilize interactions betweenCD3and Tcr chains, or alternatively, they maybe involved in signal transduction. Cytoplasmicdomainsof Ig-superfamily sequencesare mostlycompletelyunrelated, and they vary in length from 3 aminoacids for IgM(10) to 543 residues for PDGFR (46). Their roles in general are a mystery, althougha function in intracellular traffic is knownfor the cytoplasmic domainof PolyIgR(68). The unknownaspects of signal transduction are illustrated by considering Thy-1, IgM, and PDGFR, all of whichcan act as targets in the triggering of mitogenesisin various circumstances (Table 2). Thy-1 has no transmembraneprotein sequence; IgMhas such a sequencebut has almost no cytoplasmicdomain; and PDGFR has both a transmembranesequence and a very large cytoplasmicdomainwith a tyrosine kinase activity. GENETIC

LINKAGE

AND EXON STRUCTURE

It can be seen from Table 1 that loci for Ig-superfamily molecules are spread across the chromosomes.Genetic separation is common for loci that are coregulated and whose products are found in molecular complexes,but four notable cases of linkage are seen. Firstly, in all casesthe loci for V, J, andDsegmentsof antigenreceptors are linked to the C domaingenes to whichthey can be rearranged (11, 69). Presumably a relatively close cis orientation is essential for the gene rearrangement mechanism. Secondly, all the polymorphicMHC antigens are found in one large

Annual Reviews

Annu. Rev. Immunol. 1988.6:381-405. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

THE IMMUNOGLOBULIN SUPERFAMILY

395

chromosomal region, and this has beenfound in all species that havebeen investigated (16). Alsoin the mouse,the QaandT1 productsthat are very closely related to MHC class I chains are codedby loci linked to the MHC. In contrast the CD1antigens of humansare not MHC linked (21). The chains of CD1antigens are associated with fl2-m, but their sequencesare muchless closely related to class I MHC than are the Qa or T1 antigen sequences. The linkage of the polymorphicMHC antigens maybe due to selective advantagesthat result if a set of polymorphic variants favorable for antigen presentation are inherited on one chromosome. Thirdly, the two chains of the CD8antigens are closely linked to each other and to the V~loci in humanand mouse(27). Nofunctional reason for this linkage is obvious. The CD8antigens seemparticularly closely related to the V-domains of antigen receptors (56). It maybe that the and CD8genes have remainedtogether in a region of chromosome where extensive duplication occurredof genesfor heterodimerstructures related to immunity. Finally, the loci for the CD3chains, NCAM, and Thy-1are all linked on the q23 band of chromosome 11, and these loci are also linked in the mouse(15). The CD36 and ~ chain genes are arranged in reverse orientation and are separated by only 1.4 kb of sequence(70, 71). Thesegenes are within 400 kb of the CD3e gene. Thedistance betweenthe CD3genes and NCAM and Thy-1 genes is not known. There is no obvious functional reason for the chromosome 11 linkage group. Thy-1 and NCAM have in commontheir expression in neural tissues and also the fact that both moleculescan be attached to the membrane by a glycophospholipidtail. TheCD3chains are foundonly on cells in the T lymphocytelineage, but amongIg-related sequences the CD3 domains seem to match best with NCAM domains (14). Extensive gene duplication and divergenceof Ig-related sequencesmayhaveoccurred in the region nowconstituted by bandq23 of chromosome 11, and Ig-related genes nowfound in this position maybe those that have not movedto other chromosomes in evolution. Themostcharacteristic aspect of genestructure for Ig-superfamilymolecules is that the majority of the domainsequenceis often encodedwithin oneexon. This is true for all domainsin Igs andTcrs and for 20 out of 30 domainsin nonreceptor genes whosestructures are shownin Figure 3A. However, a number of examples now exist of introns found between sequencescodingfor the Cysresidues of the conserveddisulfide bonds. Thepositions of these introns in relation to the postulatedfoldingpattern of the domainsare shownin Figure 3B. Oneother exception to the one domain:one exon rule is seen in the PolyIgRwherethe domains(II) and (III) are both encodedin one exon (76). In one PolyIgRmRNA form,

Annual Reviews 396

WILLIAMS

& BARCLAY

Annu. Rev. Immunol. 1988.6:381-405. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

A. c~ ~(~O,~ll CDa~(70,71) )~tC Class I (16) ¯ (C Class II O~(72) ~qC Class II S (72)

~ LL L L L

TL (18) Qa (QT) (19,20A) ass (73) ,CD4(74) CD8(27) Thy-J (75)

L L L L L L

MRC OX-2(33) Poly Ig R (76)

L

PO (77) MAG (43)

L L

ORGANIZATION OF EXONS

00 0 0 00 00

~ D D D D D

T T TY T

D D dd D 0 D D D DD

Y

T G

Y

T T G TY

YY Y

T T

Y¥ YY

GT

YYY

o O

dd (DD) dd D D D D V dd dd dd dd dd

NCAM (40)

YY YYY

oooo

B. IG-LIKE DOMAINS ENCODED BY TWOEXONS A

B

C PO

A NCA~ DOMAIN

B C ¢ (I) (ItI)

C’ +

D ¢ (II) ¢ (IV)

C" ¢ CD4(1)

D

E F ¢ PoIyIER(I)

E

F

G

G

V-SET

Ca-SET

Figure 3 Organization of coding exons from genes for Ig-superfamily molecules excepting antigen receptor genes. A: Organization ofexons. References to each gene structure are given after the molecule name. The letters showing exon organization are coded: L, exon for leader sequence; O, an exon encoding extracellular sequence that is not Ig-related; D, an exon encoding an Ig-related domain with no introns between codons for the conserved cys residues; dd, two exons cys residues; sequence that terminus; T,

encoding an Ig-related domain with an intron between codons for the conserved (DD), an exon encoding two Ig-related domains; G, an exon for a hydrophobic is or may be cleaved from the protein when lipid is attached at the COOHan exon for a transmembrane sequence; TY, an exon for a transmembrane plus

cytoplasmic sequence; Y, an exon for cytoplasmic region sequence; -- indicates exon structure was not established. Comments: the second D in CD4 applies to the second domain in Figure 1 which will not form a standard Ig-fold but has some sequence similarities to Ig-domains (28). The exon marked G in Thy-1 is established to encode a sequence that is cleaved processing, and this is likely to be so for NCAMin which the shortest mRNAform has the exon marked G but not the TYYY exons. The longer NCAMmRNAforms splice out the G exon and include two possible combinations of the other exons. No attempt is made in this figure to show the alternative splicing events that can be seen, and noncoding exons are not shown. B. Ig-like domains encoded by two exons. The arrows indicate the positions of introns within the Ig-like domains. The letters A, B, C, C’, C", D, E, F, G indicate the positions of putative fl-strands determined by sequence similarity as indicated in Figure 2. (Data for PolyIgR from J. Harris

and K. Mostov, personal

communication.)

Annual Reviews TI4E

IMMUNOGLOBULINSUPERFAMILY

397

exon is spliced out and the mRNA is translated to give the small form of rabbit PolyIgR(76).

Annu. Rev. Immunol. 1988.6:381-405. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

FUNCTIONAL

ASPECTS

Known functions of Ig-superfamilymoleculesare given in Table 2; in all cases they include adhesionfunctions or binding functions that trigger a subsequentevent at the cell surface. A key functional feature is that homophilicor heterophilic binding occurs betweenIg-related molecules; this is often betweenmolecules on opposedmembrane surfaces. Most of the Ig-superfamilymoleculesfunction at cell surfaces; the exceptionsare antibody, the link protein, and ~lB-glycoprotein.The functions of antibodyinvolve interactions with antigen and then with effector molecules via the Fc regions to trigger subsequentevents. Thelink protein could be considered an adhesionmoleculefor binding together hyaluronicacid and proteoglycan;the function of ~ iB-gpis unknown. It remainspossible that ~B-gpis a cleaved product froma cell surface receptor in muchthe same waythat secretory componentis a product derived by proteolysis from PolylgR. In all functions in whichIg-related domainsare knownto be involved, the domaincan be consideredas providinga stable platformfor the display of specific determinantsfor recognitionreactions on the faces of B-sheets or at the bends betweenB-strands (9, 52). Thedeterminantsinvolved are likely to be mostlyprotein in nature, but there is alwaysthe possibility that the chemicalentities recognizedare carbohydratestructures.

EVOLUTION It is commonly acceptedthat all the Ig-related sequencesshownin Figure I will have been derived by gene duplication and divergence from one primordial domain. Preceding this there mayhave been a half domain structure (78) that formeda homodimer with a structure like the V-domain (79). Thehalf domainfold is postulatedto be like that of B-strandsABCC’ or GFED in Figure 2, and to associate to form a homodimer in the same waythat the ABCC’ and GFED B-strand loops associate in the V-domains (79). Thehalf-domainidea seemsto be supportedby the existence of genes that haveintrons in the midst of sequencecodingfor residues betweenthe conserveddisulfide bonds, and it is notable that in a numberof cases these comein a position that wouldroughly demarcatea half domainas

Annual Reviews

Annu. Rev. Immunol. 1988.6:381-405. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

398

WILLIAMS & BARCLAY

proposed by McLachlan (79) (Figure 3B). Genes with the intra-domain introns may be thought more directly derived from ancient genes than those lacking this feature, but this is not a reliable conclusion since the probability of intron loss is unknown.Equally unknownis the probability of intron acquisition. The possibility that the V-like fold is the most ancient is supported by the fact that Thy-1 and P0 are the only single domain molecules thought to exist without association with other chains, and both of these are in the V-SET.P0 also has an intron in the middle of the domain. If the V-fold were the most ancient type, then the C-like domain would be derived by loss of sequence from the middle of a V-type fold. A possible lineage would be V-type to C2-type to Cl-type. However, one could argue alternatively that the C2-type is the most primitive and that both V-type and Cl-type sequences were derived from this. A start from a C-type fold would not fit with the idea that the primordial domainis derived from a homodimer of half-domain structures. The Cl-type seems an unlikely candidate for the most primitive domainsince thus far Cl-set sequences have been seen only in structures associated with the immunesystem and mostly with immunerecognition. Also no intradomain introns have been seen in the Cl-set sequences. It is difficult to suggest detailed evolutionary trees for the Ig-related molecules. Somemolecules can be grouped because they show greater than average similarity within the superfamily. Somesuch groupings might be: (IgV, TcrV, CDS); (IgC, TcrC, MHC,CD1); (CD3 e, 3, ~); (CD2, 3); (MAG,NCAM); (PDGFR,CSF1R). The difficulty in trying to connect up these groups and the other molecules is that the Ig-related molecules appear to be diverging very rapidly, as assessed by the percentage of identity for equivalent chains between species. This is as low as 42%for the V-like domain of CD8chain I between rodents and humans (27) which suggests that when new genes are created they might rapidly diverge to a level where only the basic conserved patterns remain. Intermediate stages in evolution may be hard to detect in the contemporary sequences. In addition, multichain sequences have probably arisen repeatedly, and perhaps sometimes single domain structures have been rederived from multidomain forms. Examples of differences in evolution of multidomain structures can be seen with PDGFRand CSF1Rin one case and CEAin another. PDGFRand CSF1Rhave similar 5 domain patterns (46); these were probably derived from an immediate commonprecursor with the same domain pattern. In contrast, CEAhas seven domains among which the last 6 appear to have been derived by a recent double duplication of a two domainsegment (45). This is likely because within domainsII, IV, and III, V, VII there is about 70%identity, while the level of identity

Annual Reviews

Annu. Rev. Immunol. 1988.6:381-405. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

THE IMMUNOGLOBUL1N SUPERFAMILY

399

betweenthese two sets is about 25%.The nature of the immediateprecursor of CEAis thus unpredictable. In the case of rabbit PolylgR, reduction in size can be seen in the alternative splicing that producesa variant lacking domainsII and III of the structure as shownin Figure 1 (76). In terms of functional evolution the phenomenon of interactions within the superfamily(Table2) is stronglysuggestivethat the primordialfunction involveda single domaininteracting with itself probablybetweenopposed membranes.Sucha function has been suggested for the P0 myelinprotein ¯ (44). If correct, this maybe an interesting modelfor the function of the primordial domain.Heterophilic receptor pairs presumablyevolvedfrom a homophilicinteraction system, and highly specific interactions become possiblewith heterophilicrecognitionbetweendifferent cell types. It seems likely that the first functionswererelated to adhesionor triggeringat cell surfaces to control the behaviorof cells within a multicellular organism. TheIg-related moleculesthat function in neural tissues maybe mediating functions of the primitive type. Aschemefor the evolutionof heterophilic interactions froma homophilicadhesionsystembetweencells is illustrated in Figure 4A. Anotherpossibility is that heterophilic pairs giving recognitionbetween cells mayhaveoriginated fromchains that interacted on one cell to form a heterodimer(80). This is illustrated in Figure 4Bstarting froma homodimer diverging to a heterodimer and then to modified forms of the heterodimer chains interacting betweencells. The LFA-3:CD2 adhesion molecules mayhave evolved in this way from a chain that formed a homodimerand contained one Ig-related domainwith a disulfide bond and one without (NH2terminal). Fromthis type of origin the result could be a heterophilic pair, eachmember of whichis moreclosely related to the other than to other members of the superfamily(as is the case for LFA-3 and CD2)(25). If it is acceptedthat involvement in cell recognitionwasthe primaryrole of the Ig-superfamily,the question then arises--howmightthe vertebrate immunesystem be derived from this? Onepossible functional antecedent is the phenomenon of programmed cell death. In the invertebrate Caenorhabditis elegans, 25%of developingneural cells die in a predictable way, and this commonly invblves an apparentdifferentiation to cell death followedby phagocytosis(81, 82). In somecases, however,one cell appears to kill another, and this function is of the type that might be turned outwardsto producean immune system. Figure 4Csuggests that Ig-related moleculescontrol the specificity of a primitive natural killer cell and that modificationof this specificity to include determinantsof a common pathogenresulted in a killer systemto eliminatepathogen-infectedcells.

Annual Reviews V~ILLIAMS & BARCLAY

Annu. Rev. Immunol. 1988.6:381-405. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

A.

HETEROPHILIC RECOGNITION

FROM CELLS

HOMOPHILIC

ADHESION

BETWEEN

CELL

CELL 1

IG-LIKE DOMAIN STRUCTURE FOR INTERACTIONS BETWEEN PRIMITIVE CELLS (NEURAL TYPE?)

CELL

CELL 2

DUPLICATION AND DIVERGENCE TO GIVE A AND B DOMAINS SUCH THAT A:A AND A:B INTERACTIONS OCCUR BUT NOT B:B

CELL 3

DIFFERENTIAL GENE EXPRESSION SUCH THAT CELL TYPE 3 EXPRESSES ONLY DOMAIN B. CELL TYPE 3 CAN ONLY RECOGNISE TYPE

VARIOUS CELL TYPES

DUPLICATION AND DIVERGENCE OF A:B SYSTEM FOR CELL:CELL RECOGNITION AND OTHER RECEPTOR FUNCTIONS. NOTE THE AI-A n AND B1-B n UNITS MAY ALSO REPRESENT CHAINS WITH MULTIPLE DOMAINS AND TWO CHAIN STRUCTURES

VARIOUS CELL TYPES

B. HETERoPHILIC ADHESION BETWEEN CELLS FROM A HOMODIMER ON ONE CELl

C. MODIFICATION OF HETEROPHILIC RECOGNITION TO PRODUCE AN IMMUNE SYSTEM WITH SIMILARITIES TO THE VERTEBRATE T LYMPHOCYTE SYSTEM KILLER POSTULATED SYSTEM OF CELLTARGET PROGRAMMED CELL DEATH WITH SPECIFICITY CONTROLLED BY ~ ~ IG-RELATED DOMAINS INFECTED TARGET CELL ~

~

KILLER OR PHAGOCYTIC CELL

SPECIFICITY CHANGED TO INCORPORATE A DETERMINANT OF A COMMON PATHOGEN (F). DIVERSIFICATION OF THIS SYSTEM GIVES THE IG-RELATED VERTEBRATE IMMUNE SYSTEM

Figure 4 A possible schemefor structural and functional evolution in the Ig superfamily.

Annual Reviews

Annu. Rev. Immunol. 1988.6:381-405. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

THE IMMUNOGLOBULIN SUPER.FAMILY

401

Duplication and divergence of this system could lead to an immune system with the properties of the vertebrate T lymphocyte system. The B lymphocyte arm of the immune response may have evolved from this as a recognition system freed from the constraint of interaction with MHC antigens. The possibility that T cell immunity is the more primitive is supported by the finding that the T cell CD8antigen chain II has a sequence that is very like receptor J pieces without an intron or other intervening genomic sequence between the main V-like exon and the J-related piece (56). The CD8 heterodimer may be similar in its V-like domains to the structure that gave rise to the antigen receptor heterodimers. Structural and functional evolution would be greatly illuminated if invertebrate Ig-related molecules were identified. Thus far only one invertebrate sequence that might be Ig-related is known, and this is a glycoprotein of 84 amino acids with a glycophospholipid tail that was identified in a search for Thy-l-like molecules from squid neural tissue (83). The squid glycoprotein has some interesting sequence similarities to Thy1 and Ig V-domains but does not have a standard domain pattern. Thus, it cannot at this stage be added to the Ig-superfamily with the level of confidence that was applied for molecules in Figure 1. Are Ig-related structures commonin invertebrate neural cells and do invertebrate immune systems use Ig-related molecules at all? Answers to questions of this type are needed to further elucidate the structural and functional evolution of the Ig-superfamily.

ACKNOWLEDGMENTS We thank Denise Roby for help with the manuscript for photography.

and Catherine

Lee

Literature Cited 1. Hill, R. L., Delaney,R., Fellows,R. E., Lebovitz, H. E. 1966. Theevolutionary origins of the immunoglobulins.Proc. Natl. Acad. Sci. USA56:1762-69 2. Edelman, G. M. 1970. The covalent structure of a humanIgG. XI. Functional implications. Biochemistry 9: 3197-3205 3. Edmundson,A. B., Ely, K. R., Abola, E. E., Schiffer, M., Panagiotopoulos,N. 1975. Rotational allomerismand divergent evolution of domainsin Ig light chains. Biochemistry 14:395341 4. Amzel,L. M., Poljak, R. J. 1979.Threedimensionalstructure of Igs. Ann.Rev. Biochem.48:961-97

5. Peterson, P. A., Cunningham,B. A., Berggard,I., Edelman,G. M. 1972. ~2 microglobulin--afree Ig domain.Proc. NatL Acad. Sci. USA69:1697-1701 6. Becker, J. W., Reeke,G. N. Jr. 1985. Three-dimensional structure of 82microglobulin. Proc. NatL Acad. Sci. USA82:4225-29 7. Orr, H. T., Lancet, D., Robb,R. J., Lopezde Castro,J. A., Strominger,J. L. 1979. The heavy chain of humanhistocompatibility antigen HLA-B7 contains an Ig-like region. Nature282:266-70 7a. Bjorkman,P. J., Saper, M. A., Samraoui, B., Bennett,W.S., Strominger,J. L., Wiley,D. C. 1987.Structure of the

Annual Reviews

Annu. Rev. Immunol. 1988.6:381-405. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

402

WILLIAMS & BARCLAY

humanclass I histocompatibilityantigen HLA-A2. Nature. In press 8. Williams, A. F., Gagnon, J. 1982. Neuronalcell Thy-1glycoprotein: homologywith Ig. Science 216:696-703 9. Williams,A. F. 1982.Surface molecules and cell interactions. J. Theoret.Biol. 98:221-34 10. Kehry, M., Ewald, S., Douglas, R., Sibley, C., Raschke, W., Fambrough, D., Hood, L. 1980. TheIg # chains of membrane-bound and secreted IgM moleculesdiffer in their C-terminalsegments. Cell 21:393-406 11. Kronenberg,M., Siu, G., Hood,L. E., Shastri, N. 1986. Themoleculargenetics of the T-cell antigenreceptor andT-cell antigen recognition. Ann.Rev.Irnmunol. 4:529-91 12. Chien, Y.-H., Iwashima,M., Kaplan, K. B., Elliott, J. F., Davis,M.M. 1987.A newT-cell receptor genelocated within the alpha locus andexpressedearly in Tcell differentiation. Nature327:677-82 13. Weiss, A., Imboden, J., Hardy, K., Manger,B., Terhorst,C., Stobo,J. 1986. Therole of the T3/antigenreceptor complex in T-cell activation. Ann. Rev. Irnmunol. 4:593-619 14. Gold, D. P., Clevers, H., Alarcon, B., Dunlap,S., Novotny,J., Williams, A. F., Terhorst, C. 1987. Evolutionary relationship betweenthe T3 chains of the T cell receptor complexand the Ig supergenefamily. Proc.Natl. Acad.Sci. USA 84:7649-53 15. Gold, D. P., van Dongen,J. J. M., Morton,C. C., Bruns,G. A. P., van den Elsen, P., Guertsvan Kessel, A. H. M., Terhorst, C. 1987. The gene encoding the e subunit of the T3/T-cellreceptor complex maps to chromosomeI 1 in humansand to chromosome9 in mice. Proc. Natl. Acad. Sci. USA84: 166468 16. Lew,A. M., Lillehoj, E. P., Cowan,E. P., Maloy,W.L., VanSchravendijk,M. R., Coligan,J. E. 1986.ClassI genesand molecules: an update. Immunoloyy57: 3-18 17. Kaufman,J. F., Auffray, C., Korman, A. J., Shackelford,D.A., Strominger,J. 1984. The class II molecules of the human and murine major histocompatibility complex.Cell 36: 1-! 3 18. Fisher, D. A., Hunt, S. W.HI, Hood, L. 1985. Structure of a gene encodinga murine thymusleukaemia antigen, and organizationof Tla genes in the Balb/c mouse.J. Exp. Med. 162:528-45 19. Devlin,J. J., Weiss,E. H., Paulson,M., Flavell, R: A. 1985. Duplicated gene pairs andalleles of Class I genesin the

Qa2 region of the murinemajorhisto~ compatibility complex: a comparison. EMBOJ. 4:3203-7 20. Stiernberg,J., Low,M.G., Flaherty, L., Kincade, P. W. 1987. Removalof lymphocyte surface molecules with phosphatidylinositol-specific phospholipase C: Effects on mitogen responses and evidencethat ThBand certain Qaantigens are membrane-anchored via phosphatidylinositol. J. Irnmunol.138: 387784 20a. Stroynowski,I., Soloski, M., Low,M. G., Hood,L. 1987.A single geneencodes soluble and membrane-bound forms of the Qa-2antigen: anchoringof the product by a phospholipidtail. Cell 50: 759768 21. Martin, L. H., Calabi, F., Milstein, C. 1986. Isolation of CD1genes: a family of major histocompatibility complexrelated differentiation antigens. Proc. Natl. Acad. Sci. USA83:9154-58 22. Selvaraj,P., Plunkett,M.L., Dustin,M., Sanders, M.E., Shaw,S., Springer, T. A. 1987. TheT lymphocyteglycoprotein CD2binds the cell surface ligand LFA3. Nature 326:400-403 23. Brown,M. H., Gorman,P. A., Sewell, W. A., Spur, N. K., Sheer, D., Crumpton,M. J. 1987. The gene coding for the humanT lymphocyteCD2antigen is located on chromosome lp. Hum. Genet. 76:191-95 24. Williams,A. F., Barclay, A. N., Clark, S. J., Paterson,D. J., Willis, A. C. 1987. Similarities in sequencesand cellular expression between rat CD2and CD4 antigens. J. Exp. Med.165:368-80 25. Seed, B. 1987. AnLFA-3eDNAencodes a phospholipid-linked membraneprotein homologous to its receptor, CD2. Nature 329:840-42 26. Barbosa,J. A., Mentzer,S. J., Kamark, M.E., Hart, J., Strominger,J. L., Biro, P. A., Burakoff,S. J. 1985.Somaticcell hybrid analysis of humanlymphocyte function associated antigen (LFA-3): Genemappingand role in CTL-target cell interactions. LC.S.U.Short Rep. 2: 107-108 27. Littman, D. R. 1987. The structure of the CD4and CD8genes. Ann. Rev. Imrnunol.5:561-84 28. Clark,S. J., Jefferies, W.A., Barclay,A. N., Gagnon,J., William, A. F. 1987. Peptide and nucleotide sequencesof rat CD4(W3/25)antigen: evidencefor derivation froma structure with four immunoglobulin-relateddomains.Proc. Natl. Acad. Sci. USA84:1649-53 29. Johnson, P. 1987. A humanCD8(LYT3) homologueexists: Genomicsequence

Annual Reviews

Annu. Rev. Immunol. 1988.6:381-405. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

THE IMMUNOGLOBULIN SUPERFAMILY and expression, lmmuno#enetics 26: 174-77 30. Brunet,J.-F., Denizot,F., Luciani, M.F., Roux-Dosseto, M., Suzan, M., Mattei, M.-G., Goldstein, P. 1987. CTLA-4,a new memberof the immunoglobulin superfamily. Nature 328: 267-70 31. Tse, A. G. D., Barclay, A. N., Watts, A., Williams, A. F. 1985. A glycophospholipidtail at the carboxyl-terminus of the Thy-1 glycoprotein of neurons and thymocytes. Science 230: 1003-8 32. Gunter, K. C., Germain, R. N., Kroczek,R. A., Saito, T., Yokoyama, W.M., Chan,C., Weiss,A., Shevach,E. M. 1987. Thy-l-mediated T-cell activation requires co-expressionof CD3/Ti complex. Nature 326:505-7 33. McCaughan, G. W., Clark, M. J., Barclay,A. N. 1987.Characterizationof the humanhomologueof the rat MRC OX-2membraneglycoprotein. Immuno#enetics 25:329-35 34. Mostov,K. E., Friedlander, M., Blobel, G. 1984.Thereceptor for transepithelial transport of IgA and IgM contains multiple immunoglobulin-like domains. Nature 308:37-43 35. Eiffert, H., Quentin,E., Decker,J., Hillemeir, S., Hufschmidt, M., Klingmuller, D., Weber,M. H., Hilschmann,N. 1984. Die prim/irstruktur der menschlichen freien sekretkomponenteund die anordnungder disulfidbriicken. HoppeSeyler’s Zeitschrift f’dr Physioloyische Chemie365:1489-95 36. Frutiger, S., Hughes,G. J., Hanly, W. C., Kingzette, M., Jaton, J.-C. 1986. The amino-terminal domainof rabbit secretory componentis responsible for noncovalent binding to Immunoglobulin A dimers. J. Biol. Chem.261: 16673-81 37. Lewis, V. A., Koch, T., Plutner, H., Mellman, I. 1986. A complementary DNAclone for a macrophage-lymphocyte Fc receptor. Nature 324: 37275 38. Ravetch, J. V., Luster, A. D., Weinschank, R., Kochan,J., Pavlovec, A., Portnoy, D. A., Hulmes,J., Pan, Y.C. E., Unkeless,J. C. 1986. Structural heterogeneityand functional domainsof murine immunoglobulin G Fc receptors. Science 234:718-25 39. Hibbs,M.L., Classon,B. J., Walker,I. D., McKenzie,I. F. C., Hogarth,P. M. 1987. The structure of the murine Fc receptor for IgG. J. Immunol.In press 40. Cunningham,B. A., Hemperley,J. J., Murray,B. A., Prediger, E. A., Brack-

403

enbury, R., Edelman, G. M. 1987. Neuralcell adhesionmolecule:structure, Ig-like domains,cell surface modulation and alternative RNAsplicing. Science 236:799-806 41. Barthels, D., Santoni,M.-J., Wille, W., Ruppert, C., Chaix, J.-C., Hirsch, M.R., Fontecilla-Camps, J. C., Goridis, C. 1987. Isolation and nucleotide sequence of mouse NCAM eDNAthat codes for a Mr79,000polypeptide without a membrane-spanning region. EMBO J. 6: 907-14 42. Lai, C., Brow, M. A., Nave, K.-A., Noronha,A. B., Quarles, R. H., Bloom, F. E., Milner,R. J., Sutcliffe, J. G.1987. Twoforms of 1B235/myelinassociated glycoprotein, a cell adhesionmolecule for postnatal neural development,are producedby alternative splicing. Proc. Natl. Acad. Sci. USA84:4337-41 43. Salzer, J. L., Holmes,W.P., Colman,D. R. 1987. The amino acid sequences of the Myelin-associatedglycoproteins: homology to the Ig-superfamily.J. Cell. Biol. 104:957-65 44. Lemke,G., Axel, R. 1985. Isolation and sequenceof a cDNAencodingthe major structural protein of peripheral myelin. Cell 40:501-508 45. Oikawa, S., Imajo, S., Noguchi, T., Kosaki,G., Nakazato,H. 1987. Thecarcinoembryonicantigen (CEA)contains multiple immunoglobulin-likedomains. Biochem. Biophys. Res. Commun.144: 634-42 46. Yarden, Y., Escobedo, J. A., Kuang, W.-J., Yang-Feng, T. L., Daniel, T. O., Tremble,P. M., Chen, E. Y., Ando,M. E., Harkins, R. N., Francke,U., Fried, V. A., Ullrich, A., Williams,L. T. 1986. Structure of the receptor for plateletderivedgrowthfactor helps define a family of closelyrelated growthfactor receptors. Nature323:226-32 47. Sherr, C. J., Rettenmier,C. W., Sacea, R., Roussel,M.F., Look,A.T., Stanley, E. R. 1985. The c-fms Proto-oncogene productis related to the receptorfor the mononuclearphagocyte growth factor CSF-1.Cell 41:665-76 48. Ishioka, N., Takahashi,N., Putnam,F. W.1986. Aminoacid sequence of humanplasmaalphalB-glycoprotein: homology to the immunoglobulin supergene family. Proc. Natl. Acad. Sci. USA83: 2363-67 49. Bonnet, F., Perin, J.-P., Lorenzo,F., Jolles, J., Jolles, P. 1986.Anunexpected sequence homologybetweenlink proteins of the proteoglycancomplexand Ig-like proteins. Biochem.Biophys.Acta. 873:152-55

Annual Reviews

Annu. Rev. Immunol. 1988.6:381-405. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

404

WILLIAMS & BARCLAY

50. Neame, P. J., Christner,J. E., Baker,J. R. 1986. Theprimarystructure of link protein from rat chondrosarcomaproteoglycanaggregate.J. Biol. Chem.26l: 3519-35 51. McKusick,V. A. 1987. The humangene map.Genomics.In press 52. Lesk, A. M., Chothia, C. 1982. Evolution of proteinsformedby]/-sheets. II. Thecore of the Ig domains.J. MoLBioL 160:325-42 53. Amit,A.G., Mariuzza,R. A., Phillips, S. E. V., Poljak, R. J. 1986. Threedimensional structure of an antigenantibody complexat 2.8 A resolution. Science 233:747-53 54. Williams,A. F. 1987.A year in the life of the ImmunoglobulinSuperfamily. Immunol. Today 8:298-303 55. Dayhoff, M. O., Barker, W.C., Hunt, L. T. 1983. Establishing homologiesin protein sequences. Meth. Enzymol.91: 524-45 56. Johnson,P., Williams,A.F. 1986.Striking similarities betweenantigen receptor J pieces andsequencein the secondchain of the murineCD8antigen. Nature323: 74-76 57. Rudikoff, S., Pumphrey,J. G. 1986. Functionalantibodylacking a variableregion disulphide bridge. Proc. Natl. Acad. Sci. USA83:7875-78 58. Barclay, A. N., Johnson, P., McCaughan,G. W., Williams, A. F. 1987. Immunoglobulin-related structures associatedwith vertebratecell surfaces. In The T Cell Receptors. New York: Plenum.In press 59. Chatterjee, D., Maizel, J. V. Jr. 1984. Homologyof adenoviral E3 glycoprotein with HLA-DRheavy chain. Proc. Natl. Acad. Sci. USA81:6039-41 60. Huang,H.-J. S., Jones, N. H., Strominger, J. L., Herzenberg,L. A. 1987. Molecularcloning of Ly- I, a membrane glycoprotein of mouseT lymphocytes anda subsetof Bcells. Proc.Natl. Acad. Sci. USA84:204-8 61. Richardson, J. S., Richardson, D. C., Thomas,K. A., Silverton, E. W.,Davies, D. R. 1976. Similarity of three-dimensional structure betweenthe immunoglobulin domainand the copper, zinc super-oxide dismutasesubunit. J. Mol. Biol. 102:221-35 62. Maddon, P. J., Dalgleish, A. G., McDougal, J. S., Clapham,P. R., Weiss, R. A., Axel, R. 1986. The T4 gene encodes the AIDSvirus receptor andis expressed in the immunesystem and the brain. Cell 47:333-48 63. Campbell, D. G., Williams, A. F., Bayley, P. M., Reid, K. B. M. 1979.

Structural similarities betweenThy-1 antigen from rat brain and immunoglobulin. Nature282:341-42 64. Zvelebil, M. J., Barton, G. J., Taylor, W.R., Sternberg, M. J. E. 1987. Prediction of protein secondarystructure and active sites using the alignmentof homologoussequences. J. Mol. Biol. 195: 9574il 65. Campbell,R. D., Law,S. K. A., Reid, K. B. M., Sim, R. B. 1988. Structure, organization and regulation of the complementgenes. Ann. Rev. Immunol. 6:000~00 66. Parelda, R. B., Tse, A. G. D., Dwek,R. A., Williams,A. F., Rademacher,T. W. 1987. Tissue-specific N-glycosylation, site-specific oligosaccharide patterns and lentil lectin recognitionof rat Thy1. EMBO J. 6:1233-44 67. Seki, T., Chang,H.-C., Moriuchi,T., Denome, R., Ploegh,H., Silver, J. 1985. A hydrophobic transmembranesegment at the carboxylterminus of Thy-1.Science 227:649-51 68. Mostov,K. E., Kops,A. de B., Deitcher, D. L. 1986. Deletion of the cytoplasmic domain of the polymeric Immunoglobulin receptor prevents basolateral localization and endocytosis. Cell 47: 359-64 69. Wall, R., Kuehl, M. 1983. Biosynthesis and regulation of Igs. Ann. Rev. Immunol. 1:393-422 70. Tunnacliffe,A., Buluwela,L., Rabbitts, T. H. 1987. Physical linkage of three CD3genes on humanchromosome11. EMBOJ. 6:2953-57 71. Saito, H., Koyama,T., Georgopoulos, K., Clevers, H., Haser, W.G., Le Bien, T., Terhorst, C. 1987. Close linkage of the mouseand humanT3~, and ~ genes suggestsa control of their transcription by commonregulatory elements. In press 72. Trowsdale,J., Young,J. A. T., Kelly, A. P., Austin, P. J., Carson,S., Meunier, H., So, A., Erlich, H. A., Spielman,R. S., Bodmer,J., Bodmer,W. F. 1985. Structure, sequence and polymorphism in the HLA-D region. Immunol.Rev. 85: 5-43 73. Parries, J. R., Seidman, J. G.1982.Structure of wild-type and mutantfl2-microglobulin genes. Cell 29:661~59 74. Littman, D. R., Gettner, S. N. 1987. Unusual intron in the immunoglobulin domainof the newly isolated murine CD4(L3T4) gene. Nature 325:435-55 75. Gigurre, V., Isobe, K.-I., Grosveld,F. 1985. Structure of the murine Thyol gene. EMBO J. 4:2017-24 76. Deitcher, D. L., Mostov,K. E. 1986.

Annual Reviews

Annu. Rev. Immunol. 1988.6:381-405. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

THE IMMUNOGLOBULIN SUPERFAMILY Alternate splicing of rabbit polymeric Immunoglobulinreceptor. Mol. Cell. Biol. 6:2712-15 82. 77. Lemke,G., Lamar, E., Patterson, J. 1987.Isolation andanalysis of the gene encodingperipheral myelinprotein zero. Submitted 78. Bourgois, A. 1975. Evidence for an 83. ancestral immunoglobulingene coding for half a domain.Immunochemistry 12: 873-76 79. McLachlan,A. D. 1980. Early evolution 84. of the antibodydomain.In Proteins and Related Subjects, Protides of the BiolotTical Fluids, ed. H. Peeters, Pergamon 28:29-32 80. Matsunaga,T. 1985. Evolution of the 85. Ig-superfamily by duplication of complementarity. Immunol.Today6:260-63 81. Horvitz,H. R., Ellis, H. M., Sternberg, P. W. 1982. Programmed cell death in

405

nematodedevelopment.Neurosci. Commentaries 1:56-65 Hedgecock, E. M., Sulston, J. E., Thomson, J. N. 1983.Mutationsaffectingprogrammedcell deaths in the nematode Caenorhabditisele#ans. Science 220: 1277-79 Williams,A. F., Tse, A. G.-D., Gagnon, J. 1987.Squidglycoproteinswith structural similarities to Thy-1andLy-6antigens. Immuno#enetics. In press Dustin, M.L., Selvaraj, P., Mattaliano, R. J., Springer, T. A. 1987. Anchoring mechanismsfor LFA-3cell adhesion glycoprotein at membranesurface. Nature 329:846-48 Ferguson, M. A. J., Williams, A. F. 1988.Cell surface anchoringof proteins via glycosyl-phosphatidylinositol structures. Ann.Rev. Biochem.In press

NOTE ADDED IN PROOF

Additional Ig-related structures include the CD7and CD28T lymphocyte antigens, one of the chains of the mast cell Fc receptor for IgE, BLAST1, a leucocyte antigen and the proto-oncogene c-kit. CD7and CD28 antigens have a single V-like domainwith transmembranesequence and cytoplasmic domain.The CD28chain exists as a disulfide linked homodimer. In structural architecture the FcRechain is the sameas the FcR), chain, BLAST-1 is like LFA-3and CD2.Theproto-oncogenec-kit is the same as CSF1Rand PDGFR. Literature Cited Aruffo,A., Seed, B. 1987a.Molecularcloning of two CD7(T-cell leukemiaantigen) cDNAsby a COScell expression system. EMBOJ. 6:3313-16 Aruffo,A., Seed, B. 1987b.Molecularcloning of a CD28cDNA by a high efficiency COScell expression system. Proc. Natl. Acad.Sci. USA.In press Kinet, J.-P., Metzger, H., Hakimi, J., Kochan,J. 1987. A cDNApresumptively codingfor the ct subunit of the receptor with high affinity for immunoglobulin E.

Biochemistry 26:4605-10 Staunton, D. E., Thorley-Lawson,D. A. 1987. Molecularcloning of the lymphocyte activation marker Blast-1. EMBO J. 6:3695-3701 Yarden, Y., Kuang,W.-J., Yang-Feng,T., Coussens,T., Munemitsu, S., Dull, T. J., Chen,E., Schlessinger, J., Francke,U., Ulrich, A. 1987. Humanproto-oncogene c-kit: a newcell surfacereceptor tyrosine kinase for an unidentified ligand. EMBO J. 6:3341-51

Annu. Rev. Immunol. 1988.6:381-405. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Annu. Rev. Immunol. 1988.6:381-405. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Annual Reviews www.annualreviews.org/aronline Ann. Rev. [mmunoL 1988. 6: 407-38 Copyright©1988by AnnualReviewsInc..411 rights reserved

LYMPHOTOXIN Annu. Rev. Immunol. 1988.6:407-438. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Nina L. Paul and Nancy H. Ruddle Departmentof Epidemiologyand Public Health, Yale University Medical School, NewHaven, Connecticut 06510 INTRODUCTION Lymphotoxin (LT), one of the first lymphokinesto be discovered, was originally described as a correlate of the 24-hr inflammatoryresponse knownas delayed type hypersensitivity. Ruddle& Waksman noted that activated rat lymphocyteskilled syngeneicrat embryofibroblasts (1), du~ to a substance released from T lymphocytesin the presence of specific antigen (2). This phenomenon, termed"innocent bystander killing," only occurred whenlymphnode cells (LNC)from immunizedrats were cultured in the presenceof specific antigen. This inductionwasantigenspecific; the effect on target fibroblasts wasnot. Almostsimultaneously, Grangerand his colleagues described the production of a cytotoxic factor, whichthey called lymphotoxin,from murine and humanlymphocytesafter stimulation with T cell mitogens such as phytohemagglutinin(PHA)(3). The observations were later extended lymphocytesof several species including guinea pig and hamster. Theconclusion that LTproduction is an in vitro correlate of delayed type hypersensitivityis basedonthe fact that its inductiort characteristics are identical to those used to define the skin test reaction (4-7). Thus, 24-hr skin test in an animalimmunized with a haptenprotein conjugateis elicited onlyby the protein(carrier specificity). Apositiveskin test reaction can be transferred with cells (T cells), andthere is little correlation with antibody titer. LT production is elicited, only by the carrier, from T cells from rats or mice immunizedwith hapten protein conjugates. LT production was not correlated with antibody production (4-7). Furthermore,a genetic correlation existed betweenthe ability of strains of rats to manifeststrong 24-hrskin test reactions andthe ability of their T cells to produceLT(4). Early confusionregarding LTfocusedon the perception that there were 407 0732-0582/88/0410-0407502.00

Annual Reviews

Annu. Rev. Immunol. 1988.6:407-438. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

408 multiple molecular forms. Furthermore, the explosive expansion of the catalogue of lymphokinesand cytokines between1965and 1975resulted in uncertainty regarding the function and activities of any individual lymphokine. It wasoriginally suggestedthat there wereseveral size classes of LT(8) as assayedby its in vitro cytotoxicactivity against a variety of cell lines, particularly murine L929cells. However,homogeneousLTof 20-25 kd has been isolated by Aggarwalet al (9,10) from RPMI-1788,a human lymphoblastoid line, and shownto be indistinguishable from that LT designated by Granger!sgroup as ~-LT.Antibodiesprepared against this LT neutralize the cytolytic activity producedby the nonadherentcell fraction of peripheral blood leukocytes (PBL)(11), suggesting that phocytesproducea single class of LT. Theaminoacid sequenceof purified humanLT was determined and used to produce a cDNAclone (12) and to isolate the gene for humanLT (13). Wehave used this humancDNA clone to isolate the genefor murineLT(14). Someof the early uncertainty concerning LT was probably due to confusionof that lymphokine with tumornecrosis factor (TNF).Thelatter factor is frequently foundin the samesupernatantsderived fromstimulated PBLandshares almostall of LT’sbiologicalactivities. It is distinguishable. antigenically, however,and that fact and the cloning of the genes for human(15) and murine(16, 17) TNFhave allowed the resolution of problemin the clear identification of twodistinct but related molecules.It has been assumed that T cells makeLT and macrophages make TNF. Our recent analyses of T cell clones with cDNA probes and monoclonal antibodiesindicate that the cell specificity of TNFis less restricted than originally assumed.Thesimilarity in biological activity of LTand TNF has also promptedShalabyet al (18) to adopt a newnomenclaturecalling TNF,TNF-~,and LT, TNF-/3. Wethink that the previous nomenclature is moreappropriate (19); thus, in this reviewweemploythe morewidely used term, LT, which is identical to ~-LT or TNF-fl. TNFrefers to cachectin or TNF-~.Thus,the term LTidentifies that product of activated T cells that kills L929cells and is identical to the productof the cloned gene(12-14). Thebiological activities ascribed to LTmayalso be carried out by other cytokines or by LTin concert with these other molecules. Theseinclude TNF,as noted above, the interferons (IFN), interleukin (IL)-I~ and/~, natural killer cytotoxicfactor, andleukoregulin.Aneffort to establish a rational nomenclaturefor these and other cytolytic moleculesis ongoing (20). LT and TNFare clearly distinguishable from each other and from the other cytokines. The genes for TNF,IFNs, IL-ls, and LT have been cloned, and recom-

Annual Reviews

Annu. Rev. Immunol. 1988.6:407-438. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

LYMPHOTOXIN

409

binant-derivedproductstested for biological activities. That, in addition to the use of specific antibodies, has allowedthe distinction of these molecules. Furthermore,the receptors for IFNdiffer from those for ILls and for TNF/LT.Molecules whosegenes have not been cloned and whoseactivities are similar to LTinclude natural killer cytotoxic factor (21), leukoregulin, (22), and cytolytic T lymphocyte(CTL)-derived ecules LT-3(23) and CTLtoxin (24). Their distinction from LTis based ondifferencesin cells of origin, differingtarget cell sensitivity, andlack of inhibition of activity by anti-LT antibodies. Thesemoleculesneedto be better characterized to rule out their identity with LT, other cytotoxic factors, or a combinationthereof. LTis nowknownto haveseveral activities in addition to its killing of L929cells in culture. Anunderstandingof its structure, genetic organization, and regulation mayallow a better understandingof its mechanism of action anda delineation of its biological roles in defenseand disease pathogenesis.

PROTEIN STRUCTURE The amino acid sequences of murine (14, 25) and human(12) LT been determined (Figure 1A). The remarkable homology(74%) explains the lack of speciesspecificity in their biological effects. MurineLThas a 33 aminoacid signal peptide and a mature protein of 169 aminoacids. Thereis onepotential glycosylationsite at residue60 anda cysteineresidue at residue 84. Human LThas a 34 aminoacid signal peptide, a 171-amino acid matureprotein, an N-linkedglycosylationsite at residue 62, and no cysteines (12). Twoother groups have cloned and sequenced human (26, 27) and reportedan asparagineinstead of threonineat residue 26. This differencedoesnot seemto affect biologicalor antigenic characteristics of LT and maybe due to genetic polymorphism,or it could be a cloning artifact. LT isolated from RPMI-1788 has a molecular weight of 60-70 kd by gel filtration and 20 kd and 25 kd by SDS-PAGE and a pI of 5.8 (10, 28, 29). The 25 kd form is the monomericglycosylated 171 aminoacid form of LT, whichaggregates to produce the higher molecular weight forms; this explains the size heterogeneityof LTreported previously. The20 kd form is missing the first 23 aminoacids, but this does not affect the molecule’sability to kill L929cells (10,29).In vitro transcribedandtranslated murine LT cDNAproduced two sizes of LT on SDS-PAGE gel of ! 8 and 34 kd (14). Theserepresent monomeric and dimeric nonglycosylated LT forms. As recombinant E. coli-derived humanLT is active in the L929assay and neutralized by anti-LT antibodies (12), it appears that

Annual Reviews 410

PAUL & RUDDLE

Annu. Rev. Immunol. 1988.6:407-438. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

P EIRLIFIL~PIRV~C~GTIT

L H L L L L GL L LIV~LIL

P GA QGL P~GV~G

murlne human

P

human

I~ DRAFLQ DRAF

human

$SIP~LYLAHEVQL ~ITPT~I YL AHEV

QL

human

R[~Q~-~V~]L~]8 HISMY~H~GA~AIF~QIL~T

KGOQLSTHTDGI S~--~]HF~-~S[VF QGDQL 8 T HT DGI PIHLIV L~SPSITIV

Q

RQH

L

K HIV~_~A~JST

FSLSNNSLLVIPTSG~ R~)(~FS SN NSL LI

FFSSQYPF $SQYPF

L KPAAHLII

L_~DIPSKQNSL

~-~L YF

YFVYSQVVFS VYSQVVF 8(

HVPL

LSS~QK~VYPGL LS A[-~SIVYPGL

LWR

~(8C~- AY

Q~E~. QIG~"

F GAF F F GA F

Figure 1 A. Comparison of MuLTand HuL sequences. Identical amino acids are boxed. The broken lines indicate amino acid deletions in the compared sequences. This figure was previously published in (14) and is reprinted courtesy of Williams &Wilkins. B. Comparison of MuLTand MuTNF.Identical amino acids are boxed. The broken lines indicate amino acid deletions in the comparedsequences. This figure was previously published in (14) and is reprinted courtesy of Williams & Wilkins.

glycosylation is not necessaryfor at least that biological activity. It remains to be determined if and howmultimeric and differentially processed forms of LTdiffer in function. The biologically active sites of the molecule are still being determined. It is likely they reside in areas that are homologous between the two species. Alterations in the N-terminal region do not affect activity, while deletion or alteration of the C-terminal 10 (26) or 16 (12) residues abrogates LTfunction. Correspondingly, the C-terminal region of LT is highly conserved in mouse and human. The carboxyl terminal end is hydrophobic and less susceptible to proteolytic cleavage than is the amino terminus, which suggests that the N-terminal is on the exterior of the molecule and the C-terminalis interior in the native state (29). The structural relationship between LT and TNFmay reveal information concerning functional regions because the two molecules share biological activities and competefor the samereceptor (see below). Murine LT and TNF are 35% homologous with high homology concentrated in the middle and C-terminal regions (Figure 1B) (14). HumanLT and are 28%homologous and also show conservation in the C-terminus (15). The conservation of structure between murine and human LT and LT

Annu. Rev. Immunol. 1988.6:407-438. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Annual Reviews LYMPHOTOXIN 411

Annual Reviews 412

PAUL & RUDDLE

and TNFsuggests areas of conservation of function. Further structural/ functional analysis of these moleculesis necessaryto understandtheir mechanism of action and to predict their use as therapeutics for disease. In addition, the biochemicalprocessingof LT, such as the intracellular sites for glycosylation, remainsunknown.

Annu. Rev. Immunol. 1988.6:407-438. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

GENE

ORGANIZATION

AND LINKAGE

The murine LT gene has been cloned and sequenced (14, 25, 30). Its structure (shownin Figure2) consists of four exonsand three introns. The LTgene contains an octamer (TTATTTAT) 325 bases 3’ to the termination codon, first noted by Caputet al (31) in the 3’ untranslated regions several lymphokinegenes. The complementarysequence has been shown by Shaw& Kamen(32) to play a role in mRNA degradation. We(33) determined with murine-Chinesehamster somatic cell hybrids that the gene for murine LT mapsto chromosome 17 and is thus linked to H-2, the murine major histocompatibility complex (MHC).The genes for murine LT and TNFare tightly linked and are separated by only 1100 nucleotides (34). They are within the MHC between the class III and class II genes, approximately70 kb upstreamof H-2D(35). Gardnerand colleagues (25) have identified homologoussequences in the upstream region of LT, IL-2, IL-3, and granulocyte-monocytecolony-stimulating factor. Theseinteresting observationsmayprovide informationconcerning the regulation of these genes. The humanLTgene (13) is highly homologousto the murinegene (14, 30). This wouldbe anticipated by the similarities noted above in the aminoacid sequences coded by the two genes. Their homologyextends to similarities in sequence,geneorganization, linkage relationships, and chromosomalmaplocation. The humanLT gene consists of 4 exons and 3 introns; it contains the sameTTATTTAT sequence as the murine gene (31); and the distances betweenthe terminationcodonsand that sequence are similar. Someregionsin the 5’ flanking region sequencesare strikingly homologous,including the p.romoter region (W. L. Tang, S. J. Fashena, andN. H. Ruddle,in preparation),but somedifferencesexist in the introns. HumanLT maps to chromosome6 (13), within the MHC(36), and tightly linked to TNF(37). The genes for murine and humanTNFare also arranged in a way similar to those for LTand are divided into 4 exons and 3 introns. The most extensive sequence homologybetweenthe LTand TNFgenes is seen in the fourth exon(34). Thosesimilarities and the tandemarrangement the genes suggest that LTand TNFare membersofa gene family consisting of at least two members.This duplication of function and tandemarrange-

Annu. Rev. Immunol. 1988.6:407-438. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Annual Reviews LYMPHOTOXIN

413

Annual Reviews 414

PAUL & RUDDLE

ment on the chromosomeis reminiscent of the situation in the case of the genes for human IFN-a and fl, which are arranged tandemly on human chromosome9 (38). The map location of LT and TNFwithin the MHCis thus far unique for cytokines. None of the other mapped lymphokines are known to be included within a complex of genes of such profound immunologic importance.

Annu. Rev. Immunol. 1988.6:407-438. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

CELLS

OF ORIGIN

AND INDUCTION

SIGNALS

Lymphotoxinis normally a T cell lymphokine; its production is induced in functional T cells by presentation of specific antigen in an MHCrestricted fashion. Other stimuli knownto induce LT include: T cell mitogens, phorbol ester, other lymphokines, and viruses. Other cell types are knownto produce LT, including B lymphoblastoid cells and one monocytic cell line; thus far, this appears to be a characteristic of viral transformed cell lines and not of normal B cells and monocytes. T Cells T cells secrete LT after antigen or T cell mitogen stimulation, and these + are the same signals that induce T cells to proliferate. Both CD4(L3T4) and CD8(Lyt 2) + T cell subsets, when separated by antibody plus complement treatment or flow cytometry, produce LT after antigen or mitogen stimulation (6, 39-42). Somereports indicate that CD4+ T cells produce + T cells (39, 42). However,these preparations were more LT than do CD8 not entirely depleted of adherent cells, and it is possible that activated CD4+ macrophages contributed TNFto the supernatant in addition to + T cells. the LT contributed by the CD4 Our laboratory (43-46) as well as others (47) has demonstrated functional L3T4+ and Lyt-2+ murine T cell clones secrete LT in response to MHC-restricted antigen presentation. These clones can also be induced by concanavalin A (Con A) alone (43, 44). RNAhybridizing with cDNAhas been demonstrated in these T cell clones (14, 33); Leopardi Rosenau (41,48) and others (24, 49) have demonstrated LTin supernatants from human allogeneic mixed lymphocyte reactions (MLR). LT is also produced by humanCTLclones in response to antigen or Con A (24, 49). Thus, T cell receptor triggering via antigen and MHC stimulation induces LT production which can then act as an effector molecule for the T cell; mitogens bypass the need for antigenic stimulation and activate T cells in a nonspecific manner. Peripheral blood leukocytes (PBL) when stimulated with mitogen alone or with phorbol ester plus mitogen produce LT. WhenPBLare separated,

Annual Reviews

Annu. Rev. Immunol. 1988.6:407-438. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

LYMPHOTOXIN 415 more LT is produced by the nonadherentthan by the unseparated cells (11, 42, 50), and readdition of adherentcells reducesLTtiters (42). suggests the presence of possible "suppressor" factors in the adherent fractions. LTproduction evaluated by mRNA (51) and biologic assay (52) is also stimulated by the T-cell derived lymphokinesIL-2, ~-IFNplus IL-2, but not by ~-IFNalone. Thekinetics of LTproductionby PBLafter mitogen,IL-2, or phorbolesters is similar, with maximalproductionafter a 48-hr induction (42, 53). It shouldbe noted, however,that the kinetics of LT induction depends on manyfactors, including the state of cell activation before induction and the contributions of factors from other cells in a mixedpopulation. Human(54) and murine (55) LT producing T cell hybridomas allowed further study of induction signals for LT. Hybridomas mayproduceLTspontaneously(54) or in responseto T cell-specific stimuli, antiCD3antibody and mitogenplus phorbol ester (56). The induction of production in T hybridomasby antibody to CD3and alteration of LT production by antibody to Lyt-1 and Lyt-2 in an MLR (57) are analogous to the effects of antibodies to T cell surface moleculeson IL-2 production and probablymimicthe effects of antigen plus MHC. Kobayashiet al (58) have shownthat a humanT cell hybridoma(AC5-8) produces both and TNF,but that each is induced by distinct stimuli. PMAand PMA plus Con A induce LT while PMAplus the calcium ionophore A23187 induces TNF. These observations support the hypothesis that LT and TNF,though biologically similar, are inducedby distinct stimuli, and also indicate that T cells under someconditions of induction makeTNF. Other Lymphoid Cells Analysis of PBLshas shownthat normal adherent (monocyte/macrophage) populations do not produceLTand that the B cells in the nonadherent populationdo not makeLT.Stimulationof B cells with B cell rnitogens induces their proliferation but does not induce LTproduction(40). However,sometransformed B lymphoblastoidcell lines have been shown to produce LTspontaneously or after PMA induction (40, 52, 59, 60). It is fromone of these lines, RPMI 1788, that humanLTprotein wasfirst sequenced,and this led to the genecloning (12). Bersaniet al (61) shownthat antibodyto LTinhibits the cytolytic activity of Epstein-Barr virus (EBV)-transformedB lymphoblastoidcells. In addition, a human monocyticcell line generatedby transformationwith replication-defective simian virus 40 (SV40)(62) has been shownto spontaneouslyproduce L929killing factor that is neutralized with anti-LTantibody(52). This the only reported case of a macrophageproducingLT. Wehave not been able to detect production of LT mRNA or protein

Annual Reviews 416

PAUL & RUDDLE

by normal or transformed nonlymphoidcells, and this further substantiates the conceptthat LTproductionis usually T cell specific. One maypresumethat in somecases transformation of non T lymphoidcells alters the intracellular milieu in sucha waythat T cell specific genesare activated.

Annu. Rev. Immunol. 1988.6:407-438. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Virus Induction Viruses are knownto induce cytokine production, most notably IFN. EBV-immortalized and humanT cell lymphotropicvirus type I (HTLV-I)transformed cells have been shownto produce cytokines (63, 64). noted above, SV40-and EBV-transformed cell lines produce LT, though it is not clear if the virus itself is inducingLT. HTLV-Itransformed cell lines (Hut-102 and MT-2)produce LT (23, 27, 65, 66). HTLV-I mayimmortalizeT cells that are already programmed to produceLT, or it mayactivate LTvia trans-acting factors, as it has been shownthat HTLV-Ip40x induces IL-2 and IL-2R gene expression (67). It is interesting that a homology betweenthe HTLV-II long terminal repeat and the murine LT gene has been noted (25); however, these sequencesmayrepresent protein binding sites for T cell specific factors rather than viral trans-acting factors. Ruddle(68) has proposedthat the trans-acting factors of humanimmunodeficiencyvirus (HIV)mayinduce LT. To this effect, HIV-infectedPBLproducedLT activity while uninfected PBLdid not (66). Sendai virus has been shownto induce TNFbut not LT from PBL(69). LTand TNFare induced from PBLafter addition of poly IC, vesicular stomatitis virus, or herpessimplexvirus 2 (70). BIOLOGICAL

ACTIVITIES

Killing Lymphotoxin wasfirst described as a substancethat inhibited the growth of syngeneic primaryrat embryofibroblasts (71). This effect could evaluated microscopicallyor macroscopicallyand was detected as a disruption of the cell monolayer.Morphologicalchangesincluded rounding up, vacuolization, and detachmentfrom the plastic surface. It wasdeterminedthat all the detached cells were actually dead by noting their inability to excludetrypan blue dye. Alarge proportion of the remaining attached cells werealso dead. Thecytotoxic effect couldbe quantitated by counting cells in a haemocytometer or by a Coulter electronic particle counter. Numerous investigators confirmedthese results by using several different target cell lines. Theoriginal studies carried out with culture

Annual Reviews

Annu. Rev. Immunol. 1988.6:407-438. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

LYMPHOTOXIN 417 supernatants must have contained multiple lymphokines in addition to LT, most notably IFN-T(43). Thus, it is difficult in the early papers, even those with extensive biochemical purification, to determine which of the effects were due to LTalone and whichinvolved synergy with other factors. Nevertheless, most of the original experiments have now been confirmed with recombinant derived material. As noted above, the original experiments analyzing LT’s effect used rat embryo fibroblasts as targets. In subsequent published reports, syngeneic mouse embryo fibroblasts were the targets used (72). An important concept that emerged from these experiments was the realization that syngeneic fibroblasts were as susceptible as allogeneic cells. Whenit was recognized that the L929 cell, a murine fibroblast line of C3Horigin (73), could be easily and reproducibly maintained and was susceptible to LTof several species, including mouse, rat, hamster, guinea pig and human, L929 became the target of choice. There is some debate as to whether this should be considered a tumor line. It was derived from a normal mouse and does not produce tumors when injected in normal mice, though it does produce sarcomas in nude mice. If L929target cells are grownin the presence of inhibitors of protein synthesis, particularly ActinomycinD and/or cycloheximide, maximalcytotoxic effects of LT are apparent at 24 rather than 72 hr. The inclusion of such inhibitors has becomepopular, though in experiments carried out in our laboratory they have been omitted. This is so because our aims have always been to determine the biological role and the mechanism of LT killing. The inclusion of metabolic inhibitors appeared to us to conflict with those goals. Because L929cells were so frequently used as the target of LT killing, and because metabolic inhibitors were often included, the impression arose that LT was a biologically irrelevant molecule confined to a very small numberof targets. In fact, LTaffects a wide variety of cells. These effects include killing, both cytostasis and cytolysis as described in this section, and others such as the induction of differentiation described below. Target cell lines that are inhibited in addition to L929cells include the L1210 lymphoma(74). Our laboratory has noted LT inhibition of two murine macrophage cell lines, PU5.1Rand P388 D. 1, B lymphomasA20, WEHI279, 70Z-3, a myelomaP3X63NS-1,and several B cell hybridomas (75). Several T cell lymphomasare killed by LT including BW5147,YAC1, the target for NKkilling, and T cell hybridomas. P815, a mastocytoma frequently used as a target for CTLkilling, is also very susceptible to LT killing (75). It is of considerable interest that IL-2-maintained "normal" T cells are killed by LT. This occurs when LT is added to a non-LT-

Annual Reviews

Annu. Rev. Immunol. 1988.6:407-438. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

418

PAUL & RUDDLE

producing, antigen-stimulated murine T cell clone, such as 82F12 (44). has been stated that LT producing cells are somehownot susceptible to LTand arc not killed by it. Suchis not the case. If one maximallystimulates a T cell clone that can make LT and does not provide any LT absorbing targets, the T cell clone is generally killed (44). It has also been reported (75) that LTkills lipopolysaccharide-stimulated B cells. Noeffect is seen whenresting T or B cells are treated with LT. Theseresults suggest that one biological role for LT may be immunosuppression. If a T cell maximally produces LT, it may then be killed by that product and prevent the response to a particular antigen from continuing ad infinitum. The same mayoccur in the case of helper T cells. Those cells that produce IL-2 and IFN also produce LT (43). That factor could then control the B cell proliferative response induced by cells producing IL-4 and could limit that proliferation whenit was no longer needed. In several instances malignantly transformed cells are more susceptible than normal cells to LT killing. This was first reported by Meltzer & Bartlett (76) and extended by Evans and his colleagues (77) particularly in the case of cells transformed by chemical carcinogens. It is clear that rapidly proliferating cells are susceptible to LTinhibition (78). However, not all tumor cells are sensitive to LT, and it has yet to be proven that conversion to malignancyconfers susceptibility to that factor. Somecell lines are not particularly susceptible to killing by LTalone, but in the presence of minute amounts of IFN they becomevery sensitive. A synergistic effect of IFN with both human and murine LT has been noted. Williams &Bellanti (79) reported an increased killing of WI38and Hela cells by humanLT in the presence of humanIFN-~ or IFN-~. StoneWolff et al (11) noted an enhanced killing of Hela cells by humanLT the presence of IFN-7. Lee and colleagues (80) noted a similar synergy humanLT and IFN-7 in the killing of SV 40-transformed WI38cells and a synergy of murine IFN-7 with human LT in the inhibition of a B16 melanoma. These results, as noted below, can be explained in part by IFN’s ability to induce increased expression of LT receptors on some target cells. Several cell lines are not killed by LT. In a few cases this result occurs because they have very low numbers of LT receptors. In some instances the cells are not killed but are affected in other ways--either through induction of differentiation or expression of surface antigens. Somecell types are even induced to proliferate. Somecells with adequate numbers of receptors are not affected. Theseso-called null responsive cells in fact maybe affected in a mannernot yet determined. An understanding of the block in response in such cells mayprovide a clue to the mechanismof LT action.

Annual Reviews LYMPFIOTOXIN 419

Annu. Rev. Immunol. 1988.6:407-438. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Growth Stimulation TNFenhances the growth of some cell lines (81, 82). It has been assumed but not yet reported that the same is true for LT. The enhancement in growth by TNFhas usually been reported with "normal cells" such as WI38. However, as noted above, WI38becomes susceptible to LT killing in the presence of IFN (79). It is even possible that the cytotoxic agents LT and TNFcould act as growth factors at limiting dilutions while at higher concentrations they becomecytotoxic. It has not been proven that LT and TNFact on the same cellular receptor when they stimulate as when they inhibit. As noted by Sugarmanet al (81) numbers of binding sites expressed on a cell line (ME180)whose growth was inhibited were comparable to those expressed on WI38 whose growth was reported by those authors to be stimulated by TNF. Necrosis of Meth A Sarcoma In Vivo The classical assay for tumornecrosis factor is necrosis of a transplantable methyl cholanthrene-induced sarcoma (Meth A). WhenTNFis injected locally or systemically the tumor gradually undergoes necrosis and can be completely inhibited in its growth. In general one removes the tumors and evaluates them histologically for the extent of the necrosis. Both recombinant-derived human LT and LT purified from the B lymphoblastoid line RPMI1788 induced necrosis within this tumor system (12), which was indistinguishable from that induced by TNF 05). However, because the assay for necrosis of Meth A sarcoma is cumbersome, expensive, and difficult to quantitate, it is not used routinely for either LT or TNF. The mechanism of LT and TNFinduction of tumor necrosis in vivo does not appear to be due solely to direct killing because the Meth A sarcoma is not particularly LT-susceptible in vitro. Several other effects of LT noted below could contribute to the sarcoma’s necrosis, including changes in the vasculature, elicitation of cellular exudates, and increased expression of MHCantigens and subsequent CTLattack. Induction

of Antigen

Expression

LT induces or increases expression of particular antigens on several different target cells whose growth is not affected by the lymphokine. In many instances this appears to be due to the induction of a more highly differentiated state. Humanendothelial cells (HEC)are not killed by LT, but their morphologyis altered in its presence from an epithelial to a fibroblastoid form (83). There is also an increase in expression of class MHCantigens and an increase in other antigens associated with a more

Annual Reviews

Annu. Rev. Immunol. 1988.6:407-438. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

420

PAUL & RUDDLE

"differentiated" state of such cells. These include H4/18and ICAM-1, whichare associated with leukocyteadhesion.Similar effects wereseen by Collins and his colleagues (84) whotreated HECcells with TNF,though Broudyet al (85) have r6ported that LTis muchless potent than TNF inducing neutrophil adhesionmolecules. IL-I~ and IL-lfl also increased H4/18and ICAM-1 expression on HECcells. However,they did not alter MHC expression(83), and their effects wereadditive to LT’s, suggesting a different mechanism of induction of antigen expression. Class II MHC antigen expressionis increased in humanislet cells after exposure to LT or TNFin conjunction with IFN-~, (86). This is a particularly interesting observationbecauseIFN-7alone does not affect class II MHC expression on those cells. Pfizenmaier and colleagues (87) have also reported a synergistic effect of TNFandIFN-yon expressionof class II MHC antigens in two cell lines, Colo 205 and SW480. Activation of Polymorphonuclear Leukocytes Thoughpolymorphonuclearleukocytes (PMN)are usually considered endstage cells incapableof replicationor further significant differentiation, it has recently been shownthat several lymphokinesalone or in concert can enhancePMNactivities. Shalaby and colleagues (18) demonstrated that LT, TNF,and IFN-yall increase PMN’s ability to ingest latex beads, and they all enhance PMN-mediated, antibody-dependentcellular cytotoxicity (ADCC). IFNwith either LTor TNFwas moreeffective than any of the cytokinesalone. Theseresults wereconfirmedby Perussia et al (88) whoreported that LT was actually toxic to PMNat 40 units/ml but enhanced PMNphagocytic activity and ADCC at 20 units/ml. Again, synergy was seen in this system as the effects of LTwere enhancedby IFN-~,. Induction of Myeloid Leukemia Differentiation LTover the long term inhibits the growthof the promyelocyticcell lines HL-60and THP-1S (27). However,the initial responseto LTis actually increase in 3H thymidineincorporationand thus an increased proliferation (89). Furthermore,it is clear that LTdoes not actually kill the cells but induces them to differentiate to mature monocytes.After LT treatment, HL-60cells express non-specific esterase, reduce nitroblue tetrazolium, express IgG Fc receptors, and phagocytosesheep red blood cells (27). Similar effects are inducedby TNF(90) and by IFN-), in synergywith (89). Noneof the several groups that have studied this systemexamined production of TNFby the HL-60cells after treatment with LT. This is curious becauseHL-60is an excellent source of TNFafter induction with PMA or viruses (15, 70, 91). Thepossibility exists that LTinduces HL-60

Annual Reviews LYMPHOTOXIN 421 to produce TNFand that TNFitself differentiation.

Annu. Rev. Immunol. 1988.6:407-438. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Osteoclast

acts as an autocrine inducer of

Activation

LTis active in the in vitro manifestation of osteolysis. In the supernatants of humanPBLan activity defined as osteoclast activating factor (OAF) (92) was detected that stimulates resorption of bone in tissue culture. Both rLT and rTNF mimic this activity (93) as do IL-I-~ and IL-lfl (94). Furthermore, synergy between LT and either type of IL-1 occurs in this assay. Thoughthe activity was originally described as osteoclast activation, recent data suggest LTactually stimulates osteoblastic cells to release a factor whichthen stimulates osteoclasts to resorb bone (95). This is another exampleof a biological effect in which LT acts to stimulate rather than depress another cell. Antiviral Effects Both LT and TNFprotect several mouse and human cell lines against infection with virus, which include vesicular stomatitis virus, encephalomyocarditis virus, herpes simplex virus-2, and adenovirus-2 (70, 96). It interesting that two cell lines not killed by LT or TNFare protected by TNFfrom viral cytopathic effects. L929, a cell extremely sensitive to LT’s cytotoxic effects, is not protected against viral infection (96). The effects of LT and TNFon viral replication include viral protein synthesis inhibition; however, the mechanismof viral protection is not clear. Evidence conflicts concerning the involvement of IFN-%LT and TNFinduce 2’-5’(A) synthetase, a protein also induced by IFN. Furthermore, TNF induces IFN-fl2 from some cells that are protected (96, 97). However,the TNFantiviral effect was only partially inhibited by anti-IFNfl antibodies (96), and there is no strong evidence for synthesis of any other class IFN or IFN mRNA.LT probably protects against viral replication by two mechanisms--one involving IFN induction, one not. RELATIONSHIP

OF LT TO CTL

KILLING

Evidence that LTis one of the mediators of cytolytic T cell (CTL)killing and kills antigen bearing cells directly has recently been reviewed in considerable detail (98, 99). The bulk of the data are derived from our analysis of CTLclones (43-45). Such clones produce lymphotoxin and additional factors usually associated with CTLkilling including granules, perforin, and serine proteases (100-103). CTLmay be of the CD4/L3T4÷ or CD8/Lyt-2÷ phenotype, depending on class II or class I recognition, respectively. Murine LTis producedby

Annual Reviews

Annu. Rev. Immunol. 1988.6:407-438. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

422

PAUL & RUDDLE

both types of T cells (39, 43, 44) and the same L3T4+ and Lyt-2+ T cell clones that cause release of 5~CRfrom targets in a 4-hr assay also secrete LT (44, 45, 47, 104). The MHCrequirements for CTLlysis are identical to those for induction of LT production (44). Tite et al (104) found CTLclones that do not exhibit cytotoxic activity did not produce LT. Others have demonstrated that conditions that enhance LT production enhance CTLactivity (57), and inhibition of LT results in inhibition CTLactivity (45, 57). LT production by some human CD4÷ CTLclones has also been demonstrated (24, 49). Cytoplasmic granules in CTLsare associated with killing (100) and contain cytotoxic proteins (105) which mayinclude LT (46, 99). Russell colleagues (106, 107) have demonstrated that CTLcause fragmentation target cell DNA.Ucker (108) showedthat the ability of a cell to undergo DNAdegradation is necessary for a target to be lysed by a CTL. We(46, 109, 110) have shown that LT and TNFalso mediate DNAfragmentation of CTLand LT targets, including L929 cells. CTLgranules have been shownto kill L929 and cause DNAfragmentation (111); this is probably mediated by the LT (or TNF)in the granules. Argumentsagainst LT’s role in CTLkilling include the difference in respective kinetics and the absence of bystander killing. By introducing LT into target cell cytoplasm via pinocytosis and mild hypotonic shock, Schmidet al (45) showedthat LTcould cause 51Cr release from targets 4 hr. This suggests that differences seen in CTL-and LT-mediated lysis are probably due to differences in method of delivery (45). Bystander killing by antigen-specific class I and class II restricted CTLhas been shown(98, 99, 104), particularly if the experiment is carried out long enough for the specific CTLtarget to saturate its LT receptors (104). Autoreactive T cell clones also mediate bystander killing of an allogeneic bystander B 16 melanoma(112). The cells that are susceptible to bystander killing are also sensitive to LT(75). The use of anti-LT antibodies to block CTLkilling has had variable results (48, 113, 114). This conflict can now be resolved with well-characterized T cell clones and monoclonal antibodies that neutralize LT and TNF. In conclusion, the evidence is strong that LT is a major mediator of CTLkilling. Other molecules that are madeby CTLSin all likelihood also contribute to this process.

MECHANISM The wayin which LT exerts its effects on .targets is incompletely under- ¯ stood. Becauseof its pleiotropic effects on different cell types, it is important to determine whether commonmechanisms exist for LT’s cytotoxic effects on somecells and its differentiation induction of other cells. Most

Annual Reviews

Annu. Rev. Immunol. 1988.6:407-438. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

LYMPHOTOXIN

423

experimentsdesigned to provide an understanding of LT’s effects have beenconcernedwith killing, and few data have been generated regarding the mechanisms of its other effects. Several mechanisms have beenpostulated to explain LTinducedkilling. Onepossibility is that LTkills in a fashion similar to that causedby antibody and complement.If such a process occurred, the effects of LT wouldbe exerted on the target cell surface and wouldresult in a perforation. This "hole punching"wouldresult in changesin ion flow and induce colloid osmoticlysis. Alternatively, LTcould bind to a receptor. This could result in transmembranesignalling involving phosphbrylation of inactive proteins inducinga latent cellular self-destructive program.Or LT could bind to its receptor, be internalized, andact directly as a poisonto inhibit a cellular process.LT,after bindingandinternalization, couldenter and damagelysosomes,and those normallysequestered lysosomalproteins could inducesuicide. In this waythe differentiated state of the cell could influence the outcomeof the initial confrontationwith LT. Verylittle evidencesuggeststhat LTis a pore-formingprotein, similar to perforin-1 or cytolysin. Theseproteins appearto be components of ringlike or tubule structures on targets after CTLattack (101,102).LT’samino acid sequence shows no homologyto that of perforin-1 or to any complement component;there have been no ring-like or tubule structures describedon target cells after LTtreatment; and the changesin membranes are probably not profoundor early enoughto cause osmoticlysis. This section concentrates on the evidence that LT binds to a receptor. We attempt here to delineate whatoccurssubsequentto that interaction. Receptor Binding Withthe availability of recombinant-derived LT, it is possible to analyze the earliest interactions of LTwith its targets. Thedata accumulated thus far suggest that LTbinds to a receptor on target cells. Aspecific high affinity receptor for humanLT has been detected on murineL929cells (115). Recombinant-derived,nonglycosylatedhumanLThas been used demonstrate that LT and TNFcompetefor the same receptor (116) and that this receptoris different fromthe IFN-yreceptor. Becausethe majority of the workpublishedsince has involvedcharacterization of TNFbinding, those data are presented here with the caveat that the material used in most of the experiments has been humanrTNFproduced in E. coli and the conclusionsextrapolated to LT. The numberof LT(TNF)receptors calculated per cell varies whenthe samecell is studied by different investigators. Thus,L cells are reported by Rubinet al (117)to have200receptors/cell andby Tsujimotoet al (118) to have2200/ce11.Thesecalculations are influencedby the molecularweight

Annual Reviews

Annu. Rev. Immunol. 1988.6:407-438. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

424

PAUL & RUDDLE

of LT. Whenit is assumed to be biologically active as a monomerof 1625 kd, the numberof receptors is greater than 1000/cell. If one assumes that LT is normally a dimer or trimer with a molecular weight of 60,00080,000, the numberof receptors calculated is considerably smaller. Whenone studies a variety of susceptible cells, assuming an LT monomer, the numberof receptors can be as low as 1000 or as high as 15,000. The existence of a single high affinity receptor has been reported by all workers, though the affinity calculation has varied from a dissociation constant (Kd) of 6.7 × -11 (115) to x 1 0- 10(117).Neitherthe affi nity nor the number of receptors per cell can be used to distinguish between cells that are killed, stimulated, or "not affected" by TNFor LT. Thus, -1° L929 cells with approximately 2000 receptors per cell and a 1.6 x 10 M Kd are the most sensitive to killing, and RPMI7272 with 18,000 receptors per cell and a 2.3 x 10-1° Ka are resistant to TNFkilling (119), though there could be other effects of LT or TNFon RPMI7272. Someevidence suggests that the LT receptor is a glycoprotein. Several sugars, particularly those with terminal galactosyl residues, inhibit cytotoxicity of crude LT preparations (120). Furthermore, a glycoprotein 140 kd that binds to Ricinus communis agglutinin is absent in L cells resistant to LTkilling (121), although such cells were not tested for their ability to bind LT or TNF. Additional evidence for the protein nature of the receptor derives from the observation that trypsin pretreatment of sensitive cells reduces their capacity to bind radiolabeled TNF(122). Affinity cross-linking experiments of labeled TNFwith target cells have revealed a major band with a molecular weight of 95,000 (123) or 93,000 (122). This is consistent with monomerbinding to a receptor of approximately 70 kd. Recently, another TNFbinding protein of 138 kd has been detected only on cells killed by TNF,and it has been suggested that there might be a high affinity receptor whichconsists of multiple subunits (124). An increase in sensitivity of sometarget cells to LT or TNFkilling is seen after treatment with IFN, and IFN has also been reported to cause an increase in the number of TNFreceptors on target cells (116, 119). Scheurich et al (122) have argued that the enhancedLT or TNFsensitivity of IFN treated cells cannot be completely explained by an increase in receptor number or receptor affinity. For example, U937 becomes 300 times more sensitive to TNFkilling in the presence of IFN (10 ng/ml), but its receptor number and affinity are not significantly changed by this treatment. Thus, it appears that the enhanced sensitivity to LT and IFN must be explained by changes in addition to the increases in receptor number.LTand IFN-~, also synergize in effects other than killing. Changes in receptor numberor affinity have not been considered in these situations. The continued elucidation of the physical properties of the LTreceptor,

Annual Reviews LYMPHOTOXIN

425

Annu. Rev. Immunol. 1988.6:407-438. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

an understandingof its regulation, anddefinition of its ligand interactions will provide important information concerningthe effects of LTon its target cells. In those situations in whichevidenceis accumulatingthat LT contributes to the pathogenesisof the disease, an understandingof the nature of ligand receptor interaction could formthe basis for treatment. Receptor Internalization OnceLThas boundto its receptor, it is internalized and degraded.Though it binds to its receptor at 4°C, killing does not occurunless the cells are maintainedat 37°Cor higher. In fact, several workershavenoted enhanced cytotoxicity at 40°C. The temperaturedependenceof LTkilling strongly suggests a requirementfor active metabolismon the part of the target cells. Agentsthat inhibit lysosomalactivity inhibit TNF.Chloroquine does not affect TNFuptake but does decrease its degradation (118), and leupeptin, methyl amine, ammonium chloride, and chloroquineall inhibit killing by tumor necrosis serum(125, 126) or TNF(127). Pinocytosis enhancementaccelerates LTkilling. WhenSchmidand colleagues (45) subjected L929cells to a treatment that enhancedpinocytosis, the time required for LTkilling wasreducedfrom72 to 4 hr. Theworkwith lysosomotropicagents and the observationthat microinjectionof LTdirectly into cytoplasm(D. S. Schmid,N. H. Ruddle, unpublished, 127) does not result in killing together argue in favor of a pathwayother than simple uptake and toxin action. Oneof the first papers describing LT’sactivity suggested lysosmal involvementin LTmediatedkilling. Ruddle& Waksman(2) demonstratedthat treatment of target cells with LTresulted in markedincrease in staining of the lysosomal enzymeacid phosphatase. These studies do not distinguish betweenincreased synthesis of that enzymeor increased lysosomal membrane fragility and release into the cytoplasm. Membrane Chan#es The surface membranes of LTtreated cells appear remarkablyintact (127, 128). Majorstructural surface changesare seen as breaking and pinching off of membranecomponents.Thoughchanges in membranepermeability occur after LTtreatment, they do not appear so profoundas to indicate that LTkills by alterations in osmolarity.ThoughCa+ ÷ flux occurs, there is no net increase in uptake. Rosenauet al (129) reported an increase the rate of Ca+ ÷ flux betweenthe surface compartment of target cells and the intracellular compartment after LTtreatment, indicating redistribution of Ca÷ ÷ in the cell. There have been reports of activation of plasmamembranelipids and protein turnoverin L929cells after LTtreatment(130). Becausethe surface

Annual Reviews 426

PAUL & RUDDLE

membranes of LTtreated cells appear intact evenat very late times after attack, the cells maybe resisting their owndemisewith membrane repair.

Annu. Rev. Immunol. 1988.6:407-438. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Chan#es in Protein Synthesis Several effects of LT and TNFon overall protein synthesis and on the synthesis of specific proteins havebeennoted as increasedprotein turnover in L929cells (131), increased 3Hproline incorporationin bone(132), decreased expression of c-mycin HL-60cells (133). It is important determinewhetherthese changesin protein synthesis reflect attempts by the cell to repair itself or in fact contributeto the differentiationinducing effect of LT. Arole in defenseis suggestedby the observationthat treatmentof L929cells with inhibitors of protein synthesis accelerates the pace of LTkilling. Thus, a given preparation whichwould produce maximal killing at 72 hr kills at 24 hr whentargets are pretreated with Actinomycin Dor cycloheximide.This treatment does not appearto significantly affect the titer of a particular preparation,but simplythe kinetics of the reaction. It is also possible, though not proven, that LTinduces cellular toxic proteins to carry out someof its effects. TNFdoes induce production of a hematopoieticgrowthfactor from humanendothelial cells (85). Though this is one of the few activities that LT and TNFdo not share, LT mayinduce growth promotingfactors from other cells. This is further substantiated by its ability to induce an osteoclastic factor fromosteoblastic cells (95) and to increase HLA expressionby increasing both transcription and stability of HLAmRNA (86, 87). The data of Ucker(108) further implicate the target cell in its owndeath and suggest that the cytolytic processinducessynthesis of a cellular protein. In this system,a thymoma mutantis resistant to killing by glucocorticordsand by cytolytic T cells. Thesemutantcells spontaneouslyrevert to sensitivity, suggesting that restoration of a normalgenefunction confers susceptibility. Alterations

in Fatty Acid Metabolism

Phospholipids from cell membranesare converted by phospholipase Az to arachidonic acid. That in turn is convertedby cyclooxygenaseto PGG2 and by peroxidase to PHG 2 and eventually to PGE2,PGF2, and PGI2. Asthis pathwayproceeds,free radicals and reactive oxygenintermediates are generated. LT(and TNF)havebeen implicated in this pathway.Several workershavereported that both the cytotoxic and noncytotoxieeffects of LTand TNFare inhibited by aspirin and indomethacin,potent inhibitors of cyclooxygenase (134). Dayeret al (135) havereporte, d that TNFinduces PGE2synthesis in dermal fibroblasts and synovial cells. Becausethese cells are not killed by TNF,a common pathwaymayexist betweendifferent

Annual Reviews LYMPHOTOXIN

427

biological effects. The generation of free radicals, agents with profound effects on DNA,could also contribute to LT’s effects.

Annu. Rev. Immunol. 1988.6:407-438. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Induction

of DNA Fragmentation

Treatment of target cells with LT results in chromosomalchanges with fragmentation and release of target cell DNA.This occurs before cell death and is probably akin to a phenomenontermed "apoptosis" (136) postulated to be a normal cellular process which can be called into play at various stages of differentiation in which programmed cell death occurs. This appears to be the case in thymocytedifferentiation, digit formation, and resorption of the tadpole tail. Thus, one modelof LTkilling suggests that it induces a normal cellular suicide program. LT and TNFtreatment of several different target cell types including L929 fibroblasts and BW5147lymphomasresults in the release of prelabeled DNAfrom the nucleus into the cytoplasm and into the culture medium(109, 110). Electrophoretic analysis of DNAfrom LT treated cells indicates fragmentation in a regular pattern of bands that are multiples of a 180 bp subunit. These observations are consistent with the concept that breaks occur at intranucleosomal stretches of DNA.These are areas relatively unprotected by his~one proteins and thus vulnerable to attack by endonucleases. This fragmentation is an early event seen soon after LT addition, considerably before other evidence of cell death. A similar effect is seen whencytolytic T cells are addedto their specific targets. Russell and his colleagues have noted changes in nuclear membrane permeability followed by passage of nuclear DNAinto the cytoplasm and then into the culture medium(106, 107, 137). This is a very early event, occurring within minutes of CTLattack (107, 109, 137). precedes the cellular membranechanges discernible as 5~Cr release and has been likened to a process of "killing from the inside out." Antibody and complementkilling does not result in alterations of DNAstructure nor in release of prelabeled DNA.Though5 ~Cr release occurs within 30 min of addition of antibody and complement,even after 24 hr, the nuclei remain intact with no specific release of DNA.This additional difference between antibody and complement killing and the similarity with CTL killing again indicate that LTdoes not kill by colloid osmotic lysis. The mechanism of LT-induced target cell DNAfragmentation has not been determined. One hypothesis, that LT travels to the nucleus, enters through nuclear pores, and then fragments DNAseems unlikely, as LT itself does not appear to be an endonuclease(R. L. Hornung,N. H. Ruddle, unpublished). Furthermore, direct injection of TNFinto the nucleus does not kill target cells (127). LT mayactivate or cause the release of a sequestered endonuclease. The changes in lysosomal activities (2) and the

Annual Reviews

Annu. Rev. Immunol. 1988.6:407-438. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

428

PAUL & RUDDLE

importanceof lysosomal function in TNF-induced killing are consistent with this hypothesis.It is also possiblethat the changesthat occurin fatty acid metabolismand prostaglandin synthesis contribute to DNA damage. As noted above, reactive oxygenintermediates are generatedin this pathway. Such agents, knownto be extremely damagingto DNA,mayinduce the observedfragmentation.It is also possible that the integrity of the nuclear membrane is lost after LTattack, allowing access of the DNA to cytoplasmicnucleases or free radicals. Sucha changein nuclear membrane permeability occurs~very early after CTLattack (107), though nuclear membrane integrity has not been investigated after LT treatment. It is difficult to determinewhethera particular cellular changeis coincidental with LTkilling or actually contributesto that process.Docells die because their nuclei havedisintegrated and their DNA has beenreleased, or is this release a consequence of an early lytic event? It was originally stated that DNA fragmentation was not a necessary or even importantcomponent of CTLor LTkilling becauseit wasdifficult to demonstratein humancells (138). However,recent studies (139) indicate that single strand breaks do occur and can be quantitated in such cells. Certainly,target cells differ in their ability to release DNA.Therefore,that damageis probably not the most important cause of CTLor LTkilling. It will be interesting to determinewhether LT also affects the DNA of targets whichit doesnot kill but inducesto differentiate. BIOLOGICAL

ROLES

Therole of LTin vivo has alwaysbeena point of controversy.It has been consideredparadoxical that "helper" T cells should producea factor that is nonspecifically cytotoxic and capableof causing so manyalterations in cellular behavior. It is difficult to see howthat wouldenhanceantibody production. Whyshould cytotoxic T cells makea nonspecific cytotoxic factor whenthey have other moleculessuch as perforins that mightkill a cell by punchinga hole? There are several answersto these questions. It is clear that all cytokinesare antigen nonspecific.Nevertheless,production of the lymphokines,including LT, is exquisitely antigen specific and MHC restricted (44). Furthermore,other cellular mechanisms that are just beginning to be understoodensure that the moleculesare producedfo.r only a very short time (32) after antigen stimulation and that they are active locally. Thehalf life of LTand mostother lymphokines is very short, on the order of minutes, and this prevents active diffusion and widescale cytotoxicity throughoutthe body.It has beenrecently suggestedthat there ÷ class II restricted cells--those that are primarily are twoclasses of CD4 involved in B cell help and those that are most concernedwith inflam-

Annual Reviews

Annu. Rev. Immunol. 1988.6:407-438. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

LYMPHOTOXIN

429

mation (140). LT is producedby the latter cells whichalso makeIFN-y andIL-2 (43). Onecan suggest biological roles for LTfrom an analysis of in vitro activities, its activity wheninjectedin vivo, andits presenceat local reaction sites. LTprobably plays an important role in inflammationwhichcould contribute to its role in defense against tumorsand invading organisms (141). It can cause inflammationeither directly by causing tissue damage and thus eliciting a response(141) or throughits capacity to affect the vasculature as summarizedabove (83). These changes in expression leukocyteadhesionmoleculescould call forth an exudate. LT’seffects on PMN (18) also could contribute to this inflammation.Onthe basis of its knownactivities one can speculate that LTplays a role in the pathogenesis of disease, particularly someautoimmune diseases whenits normalregulation is overcome. Immunoregulation ÷ class II specific T cells and can kill B cells LTis producedby L3T4 that present antigen (104). Furthermore,LTproducingcells actually kill themselves (44). This suggests that LTmayplay an important role immunoregulation.Thoughit has not been considered a suppressor factor in the classical sense becauseit is not antigen binding, it mayact as an immunosuppressant at the local site. Defense Against Infectious

Diseases

Wehave shownthat LTis producedby cytolytic T cells, and it probably plays an important role in defense against virus infection, through its production by virus specific CTLs(142). Furthermore, LT kills virus infected cells moreeffectively than normalcells (143) and synergizes several activities withinterferon, a virus-inducedagent(11, 60, 79, 86, 89). Virus infection can induce LT production from PBL(70), and several EBV-infected long-termlines produceLTconstitutively (60, 61). ThusLT, like the interferons, maybe inducedby viruses andhaveanti-viral activity. Little evidencethus far implicatesLTin bacterial infections. It is much more likely that TNF,whichis producedby macrophagesin response to bacterial cell wall components, plays an importantrole in defense against these organisms. LTprobablyplays a role in defenseagainst parasitic infections. Malariainfected red cells induce LTproduction(142), and LTcan activate macrophages to induce killing of the schistosomula of Schistosomamansoni (144). Intriguing data implicateLTin defenseagainst chlamydiainfections (145).

Annual Reviews 430

PAUL & RUDDLE

Annu. Rev. Immunol. 1988.6:407-438. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Tumor Immunity and Graft Rejection LTprobablyparticipates in tumorimmunity.It has beenreported that it can inhibit carcinogenesisin vivo (146), andit inhibits the growthof some tumorsin vivo (12). Thetreatment of PBLwith high levels of IL-2 induces LT and TNFproduction (52). Since the same protocol is used to induce lymphokine-activated killer cells (147), LTis in all likelihood produced those cells andcontributesto their ability to affect tumorsat the sites of lymphokine-activated killer cell accumulation. LTcan be implicated in graft rejection by its production by CTL(44, 45, 104, 109). Evenmoredirect evidencederives fromits isolation from renal allografts undergoingrejection (148). In addition, the production LT is dramatically inhibited by cyclosporin A (K. M. McGrath& N. H. Ruddle,in preparation), the agentof choicein preventinggraft rejection. Disease Pathogenesis LTproduction maycontribute to tissue damageand disease pathogenesis. LTproduction reportedly occurs in such lesions as myocardialinfarcts (149), arthritic joints (150), andblisters (151). However, in suchreports is difficult to distinguish whetherthe agent is LTor TNF.LTmayplay a role in acquiredimmune deficiency syndrome(AIDS)in that it is produced by HIV-infectedPBL(66) and could contribute to their destruction (68). Arole for LTcan be speculated in autoimmune diseases. LTis produced from LNCof rats undergoing experimental allergic encephalomyelitis whenthey are exposedto myelinbasic protein, the antigen implicated in that rat modelfor multiple sclerosis (152). LTmayalso play a role diabetes. Theexpression of class II MHC gene expression on the ~ cells in the islets of Langerhansinducedby LTandIFN-y(86) could elicit CTLs whichmaycontribute to the cell destruction in this disease. LTitself may also inhibit the function of the/~ cells. LTmayalso be involvedin systemic lupus erythematosus. LT induced release of DNAfrom its targets (109, 110) mayreveal normally sequestered cellular antigens and elicit the development of antibodies to nucleic acids and histone proteins whichis so prominenta feature of this disease. Future Thereare severalapproaches that can be taken to elucidate LT’sbiological role. It should be possible to developanimalmodelsof LThypo- or hyperproduction. This has not yet occurred for LT or any other lymphokines, possibly becausesuch mutantsare lethal or becauseof the pleiotropism of cytokines. Productionof LTtransgenic mice, the availability of antibodies of LT, and the developmentof inhibitors to the LTreceptor, will

Annual Reviews LYMPHOTOXIN

431

Annu. Rev. Immunol. 1988.6:407-438. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

allow a better understanding of both its normal and pathogenic role. The availability of LT cDNAallows one to directly analyze mRNA production at the site of a particular lesion in which LTis suspected to play a role. Thus, disease lesions in whichthere is extensive lymphocyteinfiltration and proliferation, such as diabetes mellitus, multiple sclerosis, and arthritis, can be analyzed to determine if those infiltrating cells produce LT. In animal models, agents that regulate LT production or activity can be used to inhibit developmentor pathogenesis of the disease.

SUMMARY LT was one of the first lymphokines to be described and has been one of the most difficult to fit into a conceptual framework. Now,20 years after its discovery, its structure, genetic organization, and linkage are well understood in mouse and human, and insight has been gained into its biological role. It is a T cell~lerived glycoprotein of 25 kd codedby a gene within the MHC.It is somewhat (35%) structurally homologous to the macrophage product TNF. The genes for LT and TNFare tightly linked, and the proteins share most biological activities and competefor the same cell surface receptor. LT is induced in an antigen-specific MHC restricted fashion from class I and class II restricted T cells. Viral infection is also associated with LT production by lymphoidcells. LThas several effects on target cells including killing, growthstimulation, and induction of differentiation. The mechanism of LT’s effects involves receptor binding and internalization and several sequelae including changes in prostaglandins and chromosome integrity. LT probably plays several biological roles. It can contribute to immunoregulation, defense against viruses, parasitic infections, and rejection of tumors. Understanding LT’s role in the pathogenesis of diseases of autoimmunityand immunedysregulation will be the key to devising effective regimens for prophylaxis and treatment. ACKNOWLEDGMENTS

Weare grateful to Eta Kaplan for her help in manuscript preparation. This work was supported by NIH grants PO1 CA 29606 and RO1 CA 16885. N. Paul was supported by NIHTraining grant T32 AI 07019. Literature Cited 1. Ruddle, N. H., Waksman,B. H. 1967. Cytotoxic effect of lymphocyte-antigen interaction in delayed hypersensitivity. Science 157:1061~62 2. Ruddle, N. H., Waksman,B. H. 1968.

Cytotoxicity mediated by soluble antigen and lymphocytes in delayed hypersensitivity. III. Analysis of mechanism. J. Exp. Med. 128:1267-79 3. Granger, G. A., Williams, T. W. 1968.

Annual Reviews

Annu. Rev. Immunol. 1988.6:407-438. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

432

PAUL & RUDDLE

Lymphocyte cytotoxicityin vitro: Activationand release ofa cytotoxicfactor. Nature 218:1253-54 4. Ruddle, N. H., Waksman, B. H. 1968. Cytotoxicitymediatedby soluble antigen and lymphocytesin delayedhypersensitivity.II. Correlation of the in vitro responsewith skin reactivity. J. Exp. Med.128:1255--66 5. Ruddle, N. H. 1978. Delayedhypersensitivity to solubleantigensin mice. I. Analysisin oivo. Int. Archs.Aller#y Appl. Immunol. 58:56~66 6. Ruddle, N. H. 1979. Delayedhypersensitivity to solubleantigensin mice. II. Analysisin vitro, lnt. Archs.Allergy Appl. Immunol.58:44-52 7. Barry, W.B., Ruddle,N. H. 1983. Tlae delayed-typehypersensitivity response to (4-hydroxy-3-nitrophenyl) acetyl (NP) coupled proteins is carrier specific: In vivo and in vitro demonstrations. J. ImmunoL 131:70-76 8. Granger, G. A., Klostergaard, J., Yamamoto, R. S., Devlin, J., Orr, S. L., McGriff, D., Miner, K. M. 1984. Lymphotoxins--a multicomponent systemof growthinhibiting and celllytic glycoproteins. Adv, Exp. Med. Biol. 172:205-17 9. Aggarwal,B. B., Moffat, B., Harkins, R. N. 1983. Purification and eha~’acterization of lymphotoxin from humanlymphoblastoidcell line 1788. In lnterleukins, Lymphokines, and Cytokines, ed. J. J. Oppenheim, S. Cohen, pp. 521-25. NewYork: Academic 10. Aggarwal,B. B., Henzel,W.J., Moffat, B., Kohr,W.J., Harkins,R. N. 1985. Primary structure of humanlymphotoxin derived from 1788lymphoblastoid cell line. J. Biol. Chem.260: 2334-44 11. Stone-Wolff,D. S., Yip, Y. K., Kelker, H. C., Le, J., Henriksen-DeStefano, D., Rubin, B. Y., Rinderkneckt,E., Aggarwal, B. B., Vilgek, J. 1984. Interrelationship of humaninterferongammawith lymphotoxin and monocyte cytotoxin. J. Exp. Med.159: 82843 12. Gray, P. W., Aggarwal,B. B., Benton, C. V., Bringman,T. S., Henzel,W.J., Jarrett, J. A., Leung,D. W., Moffat, B., Ng,P., Svedersky,L. P., Palladino, M. A., Nedwin,G. E. 1984. Cloning and expression of the cDNAfor human lymphotoxin: A lymphokinewith tumornecrosis activity. Nature312: 721-24 13. Nedwin,G., Naylor, S. L., Sakaguchi, A. Y., Smith, D., Nedwin, G. E., Pennica,D., Goedell, D. V., Gray, P.

W. 1985. Humanlymphotoxin and tumornecrosis factor genes:structure, homology and chromosome localization. Nucleic AcidsRes. 13:6361-72 14. Li, C. B., Gray, P. W., Lin, P.-F., McGrath, K. M., Ruddle, F. H., Ruddle, N. H. 1987. Cloning and expression of murine lymphotoxin cDNA.J. Immunol. 138:4496-4501 15. Pennica, D., Nedwin,G. E., Hayftick, J. S., Seeburg,P. H., Derynek,R., Palladino, M., Kohr, W., Aggarwal,B., Goeddel, D. 1984. Human tumor necrosis factor: precursor structure, expression and homologyto lymphotoxin. Nature 312:724-29 16. Pennica,D., Hayfiick,J. S., Bringman, T. S., Palladino, M.A., Goeddel,D. V. 1985. Cloningand expression in Escherichia coli of the cDNAfor murine tumor necrosis factor. Proc. Natl. Acad. Sci. USA82:6060-64 17. Fransen, L., Muller, R., Marmenout, A., Tavernier, J., Vander Heyden,J., Kawashima,E., Chollet, A., Tizard, R., VanHewverswyn,H., VanVliet, A., Ruyssehaert,M.R., Fiers, W.1985. Molecular cloning of mouse tumour necrosis factor and its eucaryotic expression. Nucl. Acids Res. 13:4417-29 18. Shalaby, M. R., Aggarwal,B. B., Rinderkneeht,E., Svedersky,L. P., Finkle, B. S., Palladino, M. A. 1985. Activation of humanpolymorphonuclear neutrophil functions by interferon-~, and tumornecrosis factors. J. Immunol. 135:2069-73 19. Ruddle, N. H. 1986. Letter to the editor. J. Immunol.136:2335 20. Koren,H., Goldfarb, R., Henkart, P., Bleakley, C., Bonavida,B., Granger, G., Palladino, M., Podack, E., Reynolds, C., Ruddle, N., ZieglerHeitbrock,H. W.L. 1987. Proposalfor classification of leukocyte-associated cytolytic molecules.J. Leuk.Biol. 41: 447-49 21. Wright,S. C., Bonavida,B. 1987.Studies on the mechanism of natural killer cell-mediatedcytotoxicity. VII. Functional comparison of humannatural killer cytotoxic factors with recombinant lymphotoxin and tumor necrosis factor. J. lmmunol.138:1791-98 22. Ransom,J. H., Evans, C. H., McCabe, R. P., Pomato,N., Heinbaugh,J. A., Chin, M., Hanna,M. G. 1985. Leukoregulin, a direct-acting anti-cancer immunological hormonethat is distinct fromlymphotoxinand interferon. CancerRes. 45:851-62 23. Kobayashi,J., Plunkett, J. M., Masunaka, I. K., Yamamoto, R. S.,

Annual Reviews

Annu. Rev. Immunol. 1988.6:407-438. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

LYMPHOTOXIN 433 Granger, G. A. 1986. The humanLT system. XII. Purification and functional studies of LTand "TNF-like" LT forms from a continuous humanT cell line. J. lmmunol.137:1885-92 .24. Green, L. M., Reade,J. L., Ware,C. F., Devlin,P. E., Liang,C. M., Devlin, J. J. 1986.Cytotoxiclymphokines produced by cloned humancytolytic T lymphocytes. II. A novel CTL-producedcytotoxin that is antigenically distinct fromtumornecrosis factor and ~t lymphotoxin.J. Immunol.137: 348893 25. Gardner,S. M., Mock,B’. A., Hilgers, J., Huppi,K. E., Roeder,W.D. 1987. Mouse lymplaotoxin and tumor necrosis factor: Structural analysis of the cloned genes, physical linkage and chromosomal position. J. Immunol. 139:476-83 26. Kobayashi,Y., Miyamotu,D., Asada, M., Obinata, M., Osawa, T. 1986. Cloning and expression of human lymphotoxin mRNA derived from a humanT cell hybridoma.J. Biochem. 100:727-33 27. Hemmi,H., Nakamura,T., Tamura, K., Shimizu, Y., Kato, S., Miki, T., Takahashi, N., Muramatsu, M., Numao,N., Sugamura,K. 1987. Lymphotoxin: Induction of terminal differentiation of the humanmyeloidleukemiacell lines HL-60and THP-1.J. Immunol. 138:664-66 28. Aggarwal,B. B., Moffat, B., Harkins, R. N. 1984. Humanlymphotoxinproductionby a lymphoblastoidcell line, purification, and initial characterization. J. Biol. Chem.259:686-91 29. Aggarwal,B. B. 1985. Humanlymphotoxin. MethodsEnzyrnol. 116:441-48 30. Gray, P., Chen, E., Li, C. B., Tang, W. L., Ruddle, N. 1987. The murine tumor necrosis factor-beta (lymphotoxin) gene sequence. Nucleic Acids Res. 15:3937 31. Caput, D., Beutler, B., Hartog, K., Thayer,R., Brown-Shimer, S., Cerami, R. 1986. Identification of a common nucleotide sequencein the 3’ untranslated region of mRNA moleculesspecifying inflammatorymediators. Proc. Natl. Acad. Sci. USA83:1670-74 32. Shaw, G., Kamen, R. 1986. A conserved AUsequence from the 3’ untranslated region of GM-CSF mediates selective mRNA degradation. Cell 46:659-87 33. Ruddle,N. H., Li, C. B., Tang,W.L., Gray, P. W., McGrath, K. M. 1987. Lymphotoxin: Cloning, regulation, and mechanismof killing. Tumour

necrosis factor and related cytotoxins. (Ciba Found.Syrup. No. 131.) pp. 6482. Chichester:Wiley 34. Nedospasov,S. A., Hirt, B., Shakov, A. N., Dobrynin, V. N., Kawashima, E., Accolla, R. S., Jongenell, C. V. 1986.Thegenesfor tumornecrosis factor (TNF alpha) and lymphotoxin (TNF-beta)are tandemly arranged chromosome 17 of the mouse.Nucleic Acids Res. 14:7713-25 35. Muller, U., Jongenell, C. V., Nedospasov, S. A., Lindahl, K., Steinmetz, M. 1987. Tumornecrosis factor and lymphotoxingenes mapclose to H-2D in the mousemajor histocompatibility complex. Nature 325:265-67 36. Spies, T., Morton,C. M., Nedaspasov, S. A., Fiers, W.,Pious, D., Strominger, J. L. 1986. Genesfor tumor necrosis factors ~t and fl are linked to the humanmajor hlstocompatibility complex. Proc. Natl. Acad. USA83:8699v8702 37. /qedospasov, S. A., Shakhov,A. Turetskaya, R. L., Mett, V. A., Georgiev, G. P., Dobrynin,V. N., Korobko, V. G. 1985. Molecular cloning of humangenes coding for tumornecrosis factor: tandem arrangement of the alpha- and beta-genes in a short segment(16 kilobase pairs) of the human genome.Dokl. Akad. NaukUSSR286: 1487-90 38. Diaz, M.O., Lebow,M. M., Pitha, P., Rowley,J, D. 1986. Interferon and cets-1 genesin translocation(9; 11) (p22; q23) in humanacute monocyticleukemia.Science 231:265--67 39. Eardley, D., Shen, F. W., Gershon,R. K., Ruddle, N. H. 1980. Lymphotoxin production by subsets of T ceils. J. Immunol. 124:1199-1202 40. Rosenau,W., Stites, D., Jemtrud, S. 1979. Elaboration of lymphotoxinby freshly isolated humanT lymphocytes and continuous lymphoid-cell lines. Cell. Immunol.43:235-44 41. Leopardi, E., Rosenau,W.1984. Production of ~-lymphotoxinby humanT cell subsets. Cell. lmmunol.83:73-82 42. Pichyangkul, S., Miller, J. E., Waldrop, S., Khan,A. 1985. Cellular origin of human lymphotoxin and its purification. Clin. Immunol.lmmunopathol. 35:22-34 43. Conta,B. S., Powell,M.B., Ruddle,N. H. 1983. Production of lymphotoxin, IFN-~and IFN-~t, fl by murineT cell lines andclones. J. lmmunol. 130: 223135 44. Conta, B. S., Powell, M. B., Ruddle, N. H. 1985. Activation of Lyt-1÷ and Lyt-2+ T cell clonedlines: Stimulation

Annual Reviews

Annu. Rev. Immunol. 1988.6:407-438. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

434

PAUL & RUDDLE

of proliferation lymphokineproduction, andself-destruction. J. Immunol. 134:2185-90 45. Schmid, D. S., Powell, M. B., Mahoney,K. A., Ruddle, N. H. 1985. A comparisonof lysis mediatedby Lyt 2+ TNP-specificcytotoxic T lymphocyte (CTL)lines with that mediated by rapidly internalized lymphotoxincontaining supernatant fluids: Evidencefor a role of soluble mediatorsin CTL-mediated killing. Cell. Immunol. 93:68-82 46. Ruddle, N. H., McGrath,K., James, T., Schmid,D. S. 1987. Purified lymphotoxin(LT)from class I restricted CTLsand class II restricted cytolytic helpers induce target cell DNAfragmentation. In Membrane-Mediated Cytotoxicity, UCLASymposiumon Molecular Cellular Biology, ed. B. Bonavidaand R. S. Collier, 45: 379388 (NewSeries). NewYork: Liss 47. Simon,M.M., Nerz, G., Prester, M., Moll, H. 1985. Immunoregulationby mouseT cell clones. III. ClonedH-Yspecific cytotoxicT cells secrete a soluble mediator(s)that inhibits cytotoxic responsesby acting on both Lyt-2 and L3T4lymphocytes. Eur. J. Immunol. 15:773-83 48. Leopardi, E., Rosenau, W. 1982. HumanT-cell mediatedcytotoxicity: Role of subsets .and neutralization of cytotoxicity by anti-~ lymphotoxin serum. Cell. Immunol.70:148-59 49. Yamamoto, R. S., Ware, C. F., Granger, G. A. 1986. The humanLT system. XI. Identification of LTand "TNF-like"LT forms from stimulated naturalkillers, specificandnon-specific cytotoxic humanT cells in vitro. J. Immunol. 137:1878-84 50. Kelker, H. C., Oppenheim, J. D., Stone-Wolff,D., Henriksen-DeStefano, D., Aggarwal, B. B., Stevenson, H. C., Vilc, ek, J. 1985.Characterization of humantumor necrosis factor produced by peripheral blood monocytes and its separation from lymphotoxin. lnt. J. Cancer36:69-73 51. Svedersky, L. P., Nedwin, G. E., Goeddel,D. V., Palladino, M. A. 1985. Interferon-), enhances induction of lymphotoxin in recombinant interleukin 2-stimulated peripheral mononuclear cells. J. Immunol.134:1604-8 52. Nedwin, G. E, Svedersky, L. P., Bringman,T. S., Palladino, M. A., Goeddel,D. V, 1985. Effect of interleukin 2, interferon-),, andmitogenson the production of tumornecrosis factors c~ and ~. J. Immunol.135:2492-97

53. Green, L. M., Stern, M. L., Haviland, D. L., Mills, B. J., Ware,C. F. 1985. Cytotoxic lymphokines produced by cloned human cytotoxic T lymphocytes. I. Cytotoxinsproducedby antigen-specificandnatural killer-like CTL are dissimilar to classical lymphotoxins. J. Immunol.135:4034-43 54. Kobayashi, Y., Asada, M., Higuchi, M., Osawa,T. 1982. HumanT cell hybridomas producing lymphokines. I. Establishmentandcharacterizations of humanT cell hybridomas producing lymphotoxinand migration inhibiting factor. J. Immunol.128:2714-18 55. Ruddle, N. H., Conta, B. S. 1982. Lymphotoxinand immune(),) interferon production by T cell lines and hybrids. Curt. Topics Microbiol.ImmunoL 100:239-48 56. Ware, C, F., Green, L. M., Reades, J., Stern, M. L., Berger, A. E. 1986. HumanT cell hybridomas producing cytotoxic lymphokines: induction of lymphotoxinrelease and killer cell activity by anti-CD3monoclonalantibodyor lectins andphorbolester. Lymphokine Res. 5:313-24 57. Hollander,N., Shepshalovich,J. 1985. Thefunctional association of Lyt antigens with lymphokine production. Immunology54:25-33 58. Kobayashi,Y., Asada, M., Osawa,T. 1987. Production of lymphotoxinand tumournecrosis factor by a T-cell hybridoma. Immunology60:213-17 59. Granger, G. A., Moore,G. E., White, J. G., Matzinger,P., Sundsmo,J. S., Shupe, S., Kolb, W.P., Kramer,J., Glade,P. R. 1970. Productionof lymphotoxinand migrationinhibiting factor by established humanlymphocytic cell lines. J. Immunol.104:1476-85 60. Williamson, B. D., Carswell, E. A., Rubin,B. Y., Prendergast,J. S., Old,L. J. 1983. Human tumor necrosis factor producedby humanB-cell lines: synergistic cytotoxicinteraction withhu-’ maninterferon. Proc. Natl. Aead.Sci. USA80:5397-5401 61. Bersani,L., Colotta,F., Peri, G., Mantovani, A. 1987. Cytotoxic effector function of B lymphoblasts. J. Immunol. 139:645-48 62. Nagata, Y., Diamond, B., Bloom, B. R. 1983. The generation of humanmonocyte/macrophage cell lines. Nature 306:597-99 63. Lotz, M., Tsoukas, C. D., Fong, S., Dinarello, C. A., Carson, D. A., Vaughan,J. H. 1986. Release of lymphokinesafter EpsteinBarr Virusinfection in vitro. I. Sourcesof andkinetics

Annual Reviews

Annu. Rev. Immunol. 1988.6:407-438. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

LYMPHOTOXIN of productionof interferons andinterleukins in normalhumans.J. lmmunol. 136:3636-42 64. Salahuddin, S. Z., Markham,P. D., Lindner, S. G., Gootenberg, J., Popovic,M., Hemmi,H., Satin, P. S., Gallo, R. C. 1984. Lymphokineproduction by cultured humanT cells transformed by human T-cell leukemia-lymphoma virus. I. Science 223: 703-7 65. Hinuma,S., Naruo, K.-i, Tsukamoto, K., Sugamura,K., Hinuma,Y. 1985. Production of cytotoxic factor(s) humanT cell lines transformed by a humanretrovirus. Biochem.Biophys. Res. Commun.130:1052-58 66. Ratner, L., Polmar, S. H., Paul, N., Ruddle, N. 1987. Cytotoxie factors secreted by cells infected by human immunodeficiencyvirus type I. AIDS Res. Human Retroviruses. In press 67. Inoue,J. i., Seiki, M., Taniguchi,T., Tsuru, S., Yoshida,M. 1986.Induction of interleukin 2 receptor gene expression by p40x encoded by human T cell leukemia virus type I. EMBO Journal 5:2883-2888 68. Ruddle, N. H. 1986. Therole of lymphotoxin in AIDS.Immunol.Today 7: 8-9 69. Aderka,D., Holtmann,H., Toker, L., Hahn, T., Wallach, D. 1986. Tumor necrosis factor induction by Sendai virus. J. Immunol.136:2938-41 70. Wong,G. H. W., Goeddel,D. V. 1986. Tumour necrosisfactors ct andfl inhibit virus replication and synergize with interferons. Nature323:819-22 71. Ruddle, N. H., Waksman,B. H. 1986. Cytotoxicitymediatedby soluble antigen and lymphocytesin delayedhypersensitivity. I. Characterizationof the phenomenon.J. Exp. Med. 128: 123754. 72. Ruddle,N. H. 1974.Cytotoxicityreactions mediatedby antigenactivated rat and mouse lymphocytes. In Mechanismsof Cell-MediatedImmunity,ed. R. T. McCluskey,S. Cohen,pp. 4017. NewYork: Wiley 73. Sanford,K. K., Earle, W.R., Likely,G. D. 1948.Thegrowthin vitro of single isolated tissue cells. J. Natl. Cancer Inst. 9:229-46 74. Smith, M. E., Laudico, R., Papermaster,B. W.1977.Arapid quantitative assay for lymphotoxin. J. Immunol. Meth. 14:243-51 75. Powell, M. B., Conta, B. S., Ruddle, N. H. 1985.Thedifferential inhibitory effect of lymphotoxin and immune interferon on normal and malignant

435

lymphoidcells. Lymphokine Res. 4:1326 76. Meltzer, M. S., Bartlett, G. L. 1972. Cytotoxicity in vitro by products of specificallystimulatedspleencells. Susceptibility of normalcells and tumor cells. J. Natl. CancerInst. 49:1439:-43 77. Evans, C. H., Heinbaugh,J. A. 1981. Lymphotoxineytotoxieity: a combination of cytolytic andcytostatic cellular response. Immunopharmacology. 3:347-59 78. Beran, M., Anderson,B. S., Kelleher, P., Whalen,K., McCredie,K., Gutterman,J. 1987.Diversityof the effect of recombinanttumor necrosis factors alpha and beta on humanmyelogenous cell lines. Blood69:721-26 79. Williams,T. W., Bellanti, J. A. 1984. In vitro protective and enhancing effects of interferons fromseveral species on humanlymphotoxin-induced target cell killing. Cell. lmmunol.83: 25541 80. Lee, S. H., Aggarwal, B. B., Rinderknecht, E., Assissi, F., Chiu, H. 1984.Thesynergisticanti-proliferative effect of v-interferon and humanlymphotoxin. J. Immunol.133:1083-89 81. Sugarman, B. J., Aggarwal, B. B., Hass, P. I., Figari, I. S., Palladino, M.A., Shepard, H. M. 1985. Recombinant humantumor necrosis factor-alpha: effects on proliferation of normaland transformed cells in vitro. Science.230: 943-45 82. Vil~ek, J., Palombella, V. J., Hentiksen-DeStefano, D., Swenson,C., Feinmon,R~, Hirai, M., TsujimotQ,M. 1986. Fibroblast growth enhancing activity of TNFandits relationship to other polypeptide growth factors. J. Exp. Med. 163:632-43 83. Pober, J. S., Lapierre, L. A., Stolpen, A. H., Brock, T. A., Springer, T. A., Fiers, W., Bevilacqua, M. P., Mendrick, D. L., Gimbrone, M. A. 1987. Activation of cultured human endothelial cells by recombinantlymphotoxin: comparison with tumor necrosis factor and intedeukin 1 species. J. ImmunoL 138:3319-24 84. Collins, T., Lapierre,L. A., Fiers, W., Strominger,J. L., Pober, J. S. 1986. Recombinanttumor necrosis factor increases mRNA levels and surface expression of HLA-A,B antigen in vascular endothelial cells and dermal fibroblasts in vitro. Proc.Natl. Acad. Sci. USA83:446-50 85. Broudy, V. C., Harlan, J. M., Adamson, J. W.1987. Disparateeffects of tumornecrosis factor-ct/cachectin

Annual Reviews

Annu. Rev. Immunol. 1988.6:407-438. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

436

PAUL & RUDDLE

and tumor necrosis factor-]~ lymphotoxinon hematopoieticgrowthfactor production and neutrophil adhesion molecule expression by cultured humanendothelialcells. J. ImmunoL 138:4298-4302 86. Pujol-Borrell,R., Todd,I., Doshi,M., Bottazzo,G. F., Sutton, R., Gray, D., Adolf, G. R., Feldmann, M. 1987. HLAclass II induction in humanislet cells by interferon-gammaplus tumor necrosis factor on lymphotoxin.Nature 326:304-6 87. Pfizenmaier, K., Scheurieh, P., Schluter, C., Kronke,M. 1987. Tumor necrosis factor enhances HLA-A,B,C and HLA-DRgene expression in humantumor cells. J. Immunol.138: 975-80 88. Perussia, B., Kobayashi,M., Rossi, M. E., Anegon,I., Trinchieri, G. 1987. Immuneinterferon enhances function properties of humangranulocytes: Roleof Fc receptor and effect of lymphotoxin tumor necrosis factor, and granocyte-macrophagecolony stimulating factor. J. Immunol.138: 76574 89. Trinchieri, G., Kobayashi,M., Rosen, M., Loudon, R., Murphy,M., Perussia, B. 1986.Tumornecrosis factor and lymphotoxininduce differentiation of humanmyeloid cell lines in synergy with immune interferon. J. Exp. Med. 164:1206-55 90. Takeda, K., Iwamoto,S., Sugimoto, H., Takuma,T., Kawantani,N., Nodu, M., Masaki,A., Morise, H., Arimura, H., Konno,K. 1986. Identity of differentiation inducing factor and tumor necrosis factor. Nature323:338-40 91. Wang,A. M., Creasey, A. A., Ladner, M.B., Lin, L. S., Strickler, J. E., Van Arsdell, J. N., Yamamoto,R., Mark, D. F. 1985. Molecularcloning of the complementary DNA for human tumor necrosis factor. Science 228: 149-54 92. Horton, J. L., Raisz, L. G., Simmons, H. A., Oppenheim, J. J., Mergenhagen, S. S. 1972. Boneresorbing activity in supernatant fluid from cultured human peripheral blood leukocytes. Science 177:793-95 93. Bertolini, D. R., Nedwin,G. E., Bringman, T. S., Smith, D. D., Mundy,G. R. 1986. Stimulation of bone resorption and inhibition of bone formation in vitro by humantumor necrosis factor. Nature319:516-18 94. Stashenko,P., Dewhirst,F. E., Peros, W.J., Kent, R. L., Ago,J. M. 1987. Synergisticinteractions betweeninter-

leukin 1, tumor necrosis factor, and lymphotoxinin bone resorption. J. lmmunol.138:1464-68 95. Thompson, B. M., Mundy, G. R., Chambers,T. J. 1987. Tumornecrosis factor alpha and beta induce osteoblastic ceils to stimulate osteoclastic boneresorption. J. Immunol.138: 77579 96. Mestan,J., Digel, W., Mittnacht, S., Hillen, H., Blohan, D., Moiler, A., Jacobsen,H., Kirchner,N. 1986. Antiviral effects of humantumornecrosis factor in vitro. Nature323:816-19 97. Kohase,M., DeStefano-Henriksen,D., May,L. T., Vilqek,J., Setigal, P. 1986. Induction of f12 interferon by tumor necrosis factor: A homeostatic mechanismin the control of cell proliferation. Cell. 45:659-666 98. Ruddle,N. H., Schmid,D. S. 1987. The role of lymphotoxinin T-cell mediated cytotoxicity. Ann.lnst. Pasteurlmmunol. 138:314-20 99. Schmid, D. S., Ruddle, N. H. 1988. Production and function of lymphotoxinsecreted by cytolytic T cells. In Cytolytic LymphocyteClones and Complement as Effectors of the Immune System, ed. E. R. Podack.CRCPress. In press 100. Dennert, G., Podack, E. R. 1983. Cytolysisby H-2specific T killer cells: Assemblyof tubular complexeson target membranes. J. Exp. Med. 157: 1483-95 101. Henkart, P. A., Millard, P. J., Reynolds, C. W., Henkart, M. P. 1984. Cytolytic activity of purified cytoplasmic granules from cytotoxic rat large granular lymphocytetumors. J. Exp. Med. 160:75-93 102. Podack,E. R., Young,J. D. E., Cohn, Z. 1985. Isolation and biochemical functional characterization ofperforin1 fromcytolytic T-cells granules.Proc. Natl. Acad. Sci. USA82:8629-33 103. Lobe,C. G., Finlay, B. B., Paranchyck, W., Paetkau, V. H., Bleakley, R. C. 1986. Novelserine proteases encoded by twocytotoxic T lymphocyte-specific genes. Science 232:858-61 104. Tite, J. P., Powell, M.B., Ruddle,N. H. 1985. Protein-antigen specific Iarestricted cytolytic T cells: analysisof frequency,target cell susceptibility,and mechanismof cytolysis. J. Immunol. 135:25-33 105. Podack,E. R., Konigsberg,P. J. 1984. Cytolytic T cell granules: isolation, structural, biochemical,andfunctional characterization. J. Exp. Med. 160: 695-710

Annual Reviews

Annu. Rev. Immunol. 1988.6:407-438. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

LYMPHOTOXI~q 437 106. Russell, J. H., Masakowski,V. R., Dobos, C. B. 1980. Mechanismsof immunelysis. I. Physiological distinction betweentarget cell death by cytotoxic T lymphocytesand antibody plus complement. J. Immunol. 124: 1100-5 107. Russell, J. H., Dobos, C. B. 1980. Mechanisms of immune lysis. II. CTLinducednuclear disintegration of the target beginswithinminutesof cell contact. J. Immunol.125:1256q51 108. Ucker, D. S. 1987. Cytotoxic T lymphocytesand glucocorticoids activate an endogenous suicide processin target cells. Nature327:62q54 109. Schmid,D. S., Tite, J. P., Ruddle,N. H. 1986. DNAfragmentation: manifestation of target cell destruction mediatedby cytotoxicT cell lines, lymphotoxin-secreting helper T cell clones, and cell-free lymphotoxin-containing supernatants. Proc. Natl. Acad. Sci. USA 83:1881-85 110. Schmid,D. S., McGrath,K. M., Hornung, R. L., Paul, N., Ruddle, N. H. 1987. Target cell DNAfragmentation is mediatedby lymphotoxinand tumor necrosis factor. LymphokineRes. 6: 195-200 111. Konigsberg,P., Podack,E. 1986. DNA damageof target cells by cytolytic T cell granules. J. Cell Biochem.(Suppl. 10B)85 112. Shiohara,T., Ruddle,N. H., Horowitz, M., Moellmann,G. E., Lerner, A. B. 1987. Anti-tumoractivity of class II MHC antigen-restricted cloned autoreactive T cells. I. Destructionof B16 melanoma cells mediatedby bystander cytolysis in vitro. J. Immunol.138: 1971-78 113. Gately, M. K., Mayer,M.M., Henney, C. S. 1976.Effects of anti-lymphotoxin on cell-mediated cytotoxicity. Cell. Immunol.27:82-93 114. Ware, C. F., Granger, G. A. 1981. Mechanismsof lymphocyte-mediated cytotoxicity. I. Theeffects of antihumanlymphotoxinanti-sera on the cytolysis of allogeneicB cell lines by MLC-sensitizedhumanlymphocytesin vitro. J. Immunol.126:1919-26 115. Hass, P., Hotchkiss, A., Mikler, M., Aggarwal,B. 1985. Characterizationof specific high affinity receptors for humanTNFon murine fibroblasts. J. Biol. Chem.260:12214-18 116. Aggarwal,B., Eessalu, T. E., Hass,P. I. 1985. Characterizationof receptors for humantumor necrosis factor and their regulationby v-interferon. Nature 318:665~7

117. Rubin, B. Y., Anderson,S. L., Sullivan, S. A., Williamsou, B. D., Carswell, E. A., Old, L. J. 1985. Highaffinity binding of ~25I-labeled humantumor necrosisfactor (LuklI) to specific cell surface receptors. J. Exp. Med.162: 1099-1104 118. Tsujimoto,M., Yip, U. K., Vilgek, J. 1985. Tumornecrosis factor: Specific bindingandinternalization in sensitive and resistant cells. Proc. Natl. Acad. Sci. USA82:7626-30 119. Tsujimoto,M., Yip, Y. K., Vil~ek, J. 1986.Interferon-y enhancesexpression of cellular receptorsfor tumornecrosis factor. J. Immunol.136:2441-44 120. Sawada,J., Kobayashi,Y., Osawa,T. 1977.Theeffect of pulse treatment of target cells with guinea pig lymphotoxin andthe nature of its binding to target cells. Jpn. J. Exp.Med.49:93-98 121. Kobayashi,Y., Sawada,J., Osawa,T. 1979. Guinea-piglymphotoxinresistant L-cell sublines. Immunology36: 55-61 122. Scheurich, P., Ucer, U., Kronke,M., Pfizenmaier, K. 1986. Quantification and characterization of high affinity membrane receptors for tumornecrosis factor on humanleukemiacell lines. Int. J. Cancer38:127-33 123. Kull, F. C., Jacobs,S., Cuatrecasas,P. 1985.Cellularreceptorfor 125I-labeled tumornecrosis factor: Specificbinding, affinity labelingandrelationshipto sensitivity. Proc.Natl. Acad.Sci. USA82: 5756q50 124. Creasey, A. A., Yamamoto, R., Vitt, C. R. 1987. A high molecular weight component of the human tumor necrosis factor receptor is associated withcytotoxicity.Proc.Natl. Acad.Sci. USA84:3293-97 125. Kull, F. C., Cuatrecasas,P. 1981.Possible requirementof internalization in the mechanism of in vitro cytotoxicity in tumor necrosis serum. CancerRes. 41:4885-90 126. Kull, F. C., Cuatrecasas, P. 1983. Macrophage cytotoxin. In Interleukins, Lymphokinesand Cytokines, ed. J. J. Oppenheim, S. Cohen, pp. 511-19. NewYork: Academic 127. Niitsu, Y., Watanabe,N., Sone, H., Neda, N., Yamauchi,N., Urushizaki, I. 1985. Mechanism of the cytotoxic effect of tumornecrosisfactor. Jpn. J. CancerRes. 76:1193-97 128. Leopardi,E., Friend, D. S., Rosenau, W. 1984. Target cell lysis. Ultrastructural and133:3429-36 cytoskeletalalterations. J. Immunol. 129. Rosenau, W., Oie, S., Burke, G. C.

Annual Reviews

Annu. Rev. Immunol. 1988.6:407-438. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

438

PAUL & RUDDLE

1985. Calcium in lymphotoxin-mediatedcytolysis: cellularpools,fluxesand role in extracellular concentration. Cell. Immunol.95:450-57 130. Rosenau,W., Burke, G. C., Anderson, R. A. 1981. Effect of lymphotoxinon target cell plasma membranelipids. Cell. Immunol.60:144-54 131. Rosenau,W., Burke, G. C. 1982. Lymphotoxin-induced changesin target cell plasma membraneprotein. Enhancementof synthesis and content. Cell. ImmunoL67:14-22 132. Smith, D. D., Gowen,M., Mundy,J. R. 1987. Effects of interferon gamma and other cytokines on collagen synthesis in fetal rat bonecultures. Endocrinology 120:2494-99 133. Kronke,M., Schluter, C., Pfizenmaier, K. 1987.Tumornecrosis factor inhibits c-mycexpressionin HL-60cells at the level of mRNA transcription. Proc. Natl. Acad. Sci. USA84:469-73 134. Ruddle, N. H. 1987. Tumornecrosis factor andrelated cytokines. Immunol. Today 8:129-30 135. Dayer, J. M., Beutler, B., Cerami,A. 1985. Cachectin/tumornecrosis factor stimulates collagenase and prostaglandin E2 production by human synovialcells anddermalfibroblasts. J. Exp. Med. 162:216348 136. Duvafl, E., Wylie, A. H. 1986. Death and the cell. Immunol.Today7:115-19 137. Russell, J. H., Masakowski,V. R., Rucinsky,T., Phillips, G. 1982. Mechanisms of immunelysis. III. Characterization of the nature and kinetics of the cytolytic T lymphocyteinduced nuclearlesion in the target. J. lmmunol. 128:2087-94 138. Christiansen,J. E., Sears, D. W.1985. Lack of lymphocyte-induced DNA fragmentationin humantargets during lysis representsa speciesspecificdifference betweenhumanand murine cells. Proc. Natl. Acad. Sci. USA82: 448285 139. Gromkowski,S. H., Brown, T. C., Cerutti, P. A., Cerottini, J. C. 1986. DNAof humanRaji cells is damaged upon lymphocyte-mediatedlysis. J. Immunol. 136:752-56 140. Mosmann,T., Coffman, R. L. 1987. Twotypes of mousehelper T-cell cloneimplications for immuneregulation. lmmunol. Today 8:223-227 141. Ruddle, N., Homer,R. 1987. The role of lymphotoxinin inflammation.Progress AllergyIn press 142. Ruddle, N. H., Powell, M.B., Conta, B. S. 1983.Lymphotoxin, a biologically relevant model lymphokine. Lympho-

kine Res. 2:23-31 143. Aderka, D., Novick, D., Hahn, T., Fischer, D. G., Wallach, D. 1985. Increase of vulnerability to lymphotoxinin cells infected by vesicular stomatitis virus and its further augmentationby interferon. Cell. Immunol. 92:918-25 144. Esparza, I., Mannel,D., Ruppel, A., Falk, W., Krammer, P. H. 1987. Interferon ~ and lymphotoxinact synergistically to inducemacrophage killing of tumor cells and schistosomula of Schistosomamansoni.J. Exp. Med.166: 589-94 145. Byne, G. I., Kreuger,D. A. 1985. In vitro expression of factor-mediated cytotoxic activity generated during immuneresponse to Chlamydiain the mouse.J. lmmunol.134:4189-93 146. Evans, C. H., DiPaolo, J. A. 1981. Lymphotoxin: an anticarcinogenic lymphokineas measuredby inhibition of chemicalcarcinogenor ultravioletinducedtransformation of syrian hamster cells, lnt. J. Cancer27:45-49 147. Grimm,E. A., Mazumder,A., Zhong, H. Z., Rosenberg, S. A. 1982. Lymphokineactivated killer cell phenomenon:lysis of natural killer resistant fresh solid tumorcells by interleukin-2 activated autologous humanperipheral blood lymphocytes.J. Exp. Med. 155: 1823-41 148. Lowry, R. P., Marghesco, D. M., Blackburn, J. H. 1985. Immunemechanismsin organallograft rejection. VI. Delayedtype hypersensitivity and lymphotoxin in experimentalrenal allograft rejection. Transplantation40: 183-88 149. Mirrakhimov,M. M., Kitaev, M. I., Imanbaev, A. S., Markovich, M. O. 1984. Autoimmune reactions to myoglobin in myocardialinfarct patients. Ter. Arkh. 56:53-56 150. Burmester,G. R., Bech,P., Eife, R., Peter, H. H., Kalden,J. R. 1981.Induction of a lymphotoxin-likemediatorin peripheral blood and synovial fluid lymphocytesfrom patients with rheumaticarthritis. Rheumatol. lnt. 3: 13943 151. Jeffes, E. W., Yamamoto,R. S., Ahmed,A. R., Granger, G. A. 1984. Lymphotoxindetected in the blister fluid of bullouspemphigoid patients. J. Clin. Immunol.4:31-35 152. Ellison, G. W., Waksman,B. H., Ruddle, N. H. 1971. Experimental autoallergic encephalomyelitis and cellular hypersenstivityin vitro. Neurology 21:778-82

Annu. Rev. Immunol. 1988.6:407-438. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Annu. Rev. Immunol. 1988.6:407-438. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Annual Reviews www.annualreviews.org/aronline Ann. Rev. Immunol.1988.6: 439~54 Copyright© 1988by AnnualReviewsInc. All rights reserved

Annu. Rev. Immunol. 1988.6:439-464. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

REGULATION OF CYTOKINE GENE EXPRESSION Tadatsugu Taniguchi Institute for Molecularand Cellular Biology,OsakaUniversity, Suita-shi, Osaka 565 Japan INTRODUCTION Cytokinesrepresent essential soluble transmitters of cell-to-cell communication and play crucial roles in manybiological processes; these include viral infection, inflammation,immunity,and hematopoiesis.The term lymphokinewas first introduced in 1969, following discovery of a variety of factors producedby mitogenor antigen-activated lymphocytes that affect the growthandmobilityof leukocytes(1). Subsequently,it was realized that factors similar in function were also producedby nonlymphocyticcells such as macrophages,keratinocytes, fibroblasts, and transformedcell lines. Thus, those factors as a class have beentermed cytokines (2). Among the cytokines, factors producedby monocytessuch as macrophagesare sometimesreferred to as monokines.Evidence has also accumulatedthat someof the knowncytokinesfunction in the nervous system(3). Withthe advent of recombinantDNAtechnology, manyof the cytokine genes have been isolated, and their products have becomeavailable in pure form. This molecularapproachhas shed light on heretofore unclear structure-function relationships of the cytokines. Mostof the cytokines indeedmanifestmultiplebiologicalactivities on different target cells, and manycytokines are producedby a variety of cell types in response to different stimuli. Furthermore,the expressionof a given cytokinegeneis invariably influenced by other cytokines, forminga networkof "cytokine cascades"(4, 5). Suchcascadesmayrepresent a salient feature of cytokine action in the programmed growth,differentiation, and function of cells. Thus, the expressionof cytokinegenes appears to be regulated tightly by complexmechanisms; it is quite conceivablethat dysregulationofcytokine 439 0732-0582/88/0410-0439502.00

Annual Reviews

Annu. Rev. Immunol. 1988.6:439-464. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

440

TANIGUCHI

genes (or cytokine receptor genes) maycontribute to the development various diseases including neoplasia. RecombinantDNA technology has provided the opportunity for cloning and characterizing various cytokine genes and for gaining insights into the mechanismsby whichcytokine genes are inducedin response to extracellular stimuli in certain cell types. Theavailability of such genes madeit possible (a) to analyze quantitative changesin cytokine-specific mRNA levels under various conditions, (b) to elucidate the structure the DNA segmentspossibly involved in the regulation of cytokine genes, (c) to identify functional cis-acting DNA sequences required for gene regulation by using gene transfer techniques, and (d) to identify transacting regulatoryfactors. At present, however,information available about the molecular mechanismsregulatingthe expressionof cytokinegenesis limited. In this article I summarizethe currently accumulatedinformation on the regulation of cytokinegene expression. REGULATION EXPRESSION

OF THE CYTOKINE GENE AT THE TRANSCRIPTIONAL

LEVEL

Except in sometransformed cells, most of the cytokine specific mRNAs are usually undetectableunless the cells are inducedby the properstimuli. Theexpression of these genes is apparently controlled primarily at the transcriptional level. Notethat, as describedbelow,inductionof the transcription of manycytokine genes does not require de novo protein synthesis.This impliesthat the extracellularstimulationof cells byinducers results in activation of otherwisenon-operativetranscriptional machinery throughmodificationof preexistingfactors (either repressorsor activators) involvedin transcription. Virus-Inducible Interferon Genes (Type I Interferon Genes) Three types of interferons (IFNs) (proteins with antiviral activity) been described in humans,and their genes have been analyzed in detail (6-8). Among them, the genes for IFN-~and IFN-/3, but not for IFN-y, can be inducedas a result of a viral infection. BothIFN-~and IFN-//are classified as type I IFNs; IFN-y, a lymphokineproducedby T cells, is classified as a type II IFN(6, 7). TypeI IFNsare encodedby a superfamily including the IFN-~family, whichis multimembered in humansas well as in other species, and the IFN-//family, whichhas manymembers in cattle but only one in humansand mostother species (8). The synthesis of IFN0~ and IFN-/~is not detectable in normallygrowingcells but reaches high

Annual Reviews CYTOKINE GENE REGULATION

441

levels after induction by a virus or by double-stranded RNAssuch as poly(rI) : poly(rC). Evidence also suggests the induction of these IFNs other cytokines such as platelet-derived growth factor (PDGF)(9), colony stimulating factor-1 (CSF-1) (10, 11), and interleukin-1 (IL-1) (12). expression of IFN-~ and IFN-fl is tissue specific in humans: leukocytes (monocytes) express IFN-~ predominantly, whereas, with some exceptions, fibroblasts exclusively express IFN-fl. Besidetheir antiviral activity, multiple biological activities have been ascribed to both 0~ and fl IFNs (6Annu. Rev. Immunol. 1988.6:439-464. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

8). Induction of the IFN genes comes about by the activation of transcription. Using nuclear transcription assays, Raji & Pitha have shown that the synthesis of IFN-fl-specific RNAin nuclei becomes detectable after induction of a humanfibroblast cell line by poly(rI) : poly(rC) (13). For both IFN-~ and IFN-fl genes activation of mRNA transcription is mediated by the Y-flanking sequences of the genes (14, 15), which show some degree of homologyto each other (Figure 1) (8, 16). By creating various deletions in the 5’-flanking region of the genes and expressing the resulting mutant genes in various host cells, several groups have identified the DNAsequences responsible for induction-specific transcription of the genes. By stably introducing various deletion mutants of the IFN-~ 1 gene into mouseL929cells, Ragget al located the 5’ boundaryof the region required for the virus-mediated induction of transcription between positions - 117 and -74 with respect to the cap site (17). Furthermore, Ryals et

IFN-~ GENE IFN-~VRE re~ion I -110

-100

-g~

-80

-70

I

-~0

-50

-40

-~0

AAGAGT C~AT~A ¯ ..ACAA A AGCAAA AA..CA 6 ~]A[~CCCAGAAGCATTAA..GAAAGTGGAAATCAGTA TGTTCCC TATTT~ A ]AAATGTJJAAATGAJ C r~ A ~’~ ~’~’~’~ G ~’~. i’~ OAAATTCCTCTGAATAGAGAGAGGACCATCTCA ....... TAT~ I

constitutive element ,~

I

negative element

I

/ TATA box

inducible elements

IFN-/~ GENE 1 TheregulatoryDNA sequenceswithin the humanIFN-~tand IFN-fl genes. The repetitive hexameric sequencesare framed.Gapsare introducedin order to maximize the sequence homology between the twogenes.Seetext andRefs.18, 24, 27, 33, 34, 35for the detailsof the regulatory sequence elements. Figure

Annual Reviews

Annu. Rev. Immunol. 1988.6:439-464. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

442 TANIGUCHI identified a 46-base pair (bp) virus responsive element (VRE)within Y-flanking region (sequences from -109 to -64) of the human IFN-al gene (18) (Figure 1). Whenthis DNAsegment was linked adjacent to heterologous rabbit fl globin gene promoter, it made the promoter virusinducible. Because the basal promoter activity of the globin gene was not diminished by placing the IFN-~I promoter sequences in various positions, the induction was attributed mainly to positive rather than to negative control (18). Other IFN-a genes are also induced by viruses, but there is a striking difference in the level of the individual IFN-~t mRNA species induced in different cell types. Moreover,this difference cannot be attributed to differences in the induction protocol (19). These observations suggest that there maybe different mechanismsregulating transcriptional induction pathways and/or differential posttranscriptional control of the IFN mRNAs in various cell types. Deletion analyses were also carried out by a number of groups for the human IFN-fl gene, but the 5’ boundaries for the maximal induction identified differed from one group to the other (20-25). In this regard, is worth noting that induction-dependent stabilization of IFN-fl mRNA has been documented(13, 26). The existence of such a posttranscriptional control mechanism may sometimes lead to a mistake in identifying the essential control region of transcription, particularly if one monitors the accumulated level of the IFN-fl activity to measure the extent of induced transcription. The regulatory DNAsequences identified may turn out to be variable in some cases depending on which of various host-vector systems is used. It is therefore important to assess the function of given regulatory sequences by using different host-vector systems. Using a stable transformation procedure similar to that used by Ryals et al (18) for the IFN-al gene, Fujita et al (24) showedthat the 5’ boundary of the IFN-fl gene required for the maximalinduction lies between - 117 and - 105 in mouseL929cells. These results were in agreement with those of Dinter et al (25) and were further supported by results of using transient expression system (27). The authors observed that the level induction of both human IFN-fl mRNAand IFN-fl activity in L929 cells by Newcastle disease virus (NDV)diminished dramatically as the deletion extended from -105 to -93 (24). Essentially the same results were obtained by using different inducers such as poly(rI) : poly(rC) Sendai virus (24, 27). On the other hand, Zinn et al (23) and Goodbourn et al (29) concludedthat the critical 5’ boundaryfor induction lies between -79 and -75 (-77 and -73 by their numbering) in mouse C127 cells transformed by bovine papilloma virus-derived vectors carrying the various IFN-fl deletion mutants. They obtained essentially similar results by using another host-vector system, except in Hela cells where they found a

Annual Reviews

Annu. Rev. Immunol. 1988.6:439-464. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

CYTOKINE

GENE

REGULATION

443

sequence requirement similar to that reported by Fujita et al (24) and Dinter et al (30). Also, in a transient expression system using the C127 cells, Goodburnet al found that the deletion to -79 did not abolish the inducibility of the gene but that the additional sequence up to - 109 (i.e. - 107 by their numbering) was required for the maximalinduction (29). The different sequence requirements for the maximalinduction of the IFNfl gene maybe explained at least in part by assumingthat cellular factor(s) interacting with the regulatory DNAsequencesis present either in different levels or in a different form (or both) depending on cell types or on the state of cells. In other words, the sequence requirement may depend on the state of the cellular factor(s). Evidencefor the presenceof the positive regulatory factor(s) has been presented by Fujita et al (27). The identified regulatory DNAsequences of the human IFN-fl gene were shownto function as a virus-inducible enhancer (24, 29). The region basically consists of repeated hexanucleotide sequences (7 times) between -109 and -65, where the consensus sequence was deduced as A-T A-A-~.~-GA (25) (Figure 1). In fact, when chemically synthesized sensus sequences were used, most of such sequence units were shown to mediate virus-induced activation of transcription whenthey were tandemly repeated (27). The incremental contribution of such a hexanucleotide unit in virus-inducible enhancer function supports the notion that such sequence motifs within the essential regulatory region of the IFN-fl gene function cooperatively and contribute in the induction presumably by interacting with a positive regulatory fact0r(s ). Indeed, introduction of point mutations within the sequence motifs located either upstream (- 109 to -98) or downstream (-76 to -65) of the gene resulted in dramatic reduction of inducibility (31). A somewhatsimilar observation has also been made by Dinter & Hauser, who showed that duplication of the sequence between -92 and -53 (which contains four blocks of "the hexamerunit") leads to a severalfold increase in the expression of IFN-fl (32). They also reported the presence of regulatory sequences downstream of the TATApromoter sequence of the gene. Subsequences within the regulatory region (VRE)of the humanIFN-~I gene also appear to confer virus-inducibility whenthey are multimerized (33). A series of studies by Maniatis and his colleagues led them to propose that the enhancer of the IFN-fl gene consists of a "constitutive transcription element" and a "negative regulatory element" that blocks an otherwise functional, constitutive enhancerprior to induction (29, 34, 35). Evidencewas also provided for the existence of a repressor that is boundto the negative clement, located immediately downstreamof the constitutive element (35). A sequence element that mimics such a negative element has not been identified in the IFN-c~ genes. The authors showed that the

Annual Reviews

Annu. Rev. Immunol. 1988.6:439-464. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

444

TANIGUCHI

constitutive element activates the distal promoter sequence of the gene for Herpes simplex virus thymidine kinase (HSV-TK)when it is dissociated from the downstreamnegative element (34). At present, their results and the results of Fujita et al (27) are not easily reconciled: The "constitutive element" contains the abovementionedhexanucleotide motifs that mediate inducibility by virus but have no constitutive activity per se (27). One explanation may be that, in addition to the hexamer motifs, a sequence element resembling the SV40enhancer core sequence (-68 to -61) constitutes the constitutive enhancer described by Goodbournet al (34). Thus, transcription of the IFN-fl gene might be regulated by three elements: (a) inducible elements (27), (b) a constitutive element whosefunction requires two of the inducible elements (therefore, the constitutive element per se mayalso be inducible) (34), and (c) a negative element (Figure 1) (34, Taken together, the results presented by various groups raise the question as to what extent such elements each contribute to virus-induced transcription; is it activation or derepression or both that mainly determine the induction level? Fujita et al found that the SV40enhancer placed just upstream of the repeated hexameric sequence, (A-A-G-T-G-A)× 4, failed to exert its function on distal chloramphenicol acetyltransferase (CAT) gene expression unless the recipient cells were virus-induced (27, 31). This "repressing" or "silencing" effect has also been observed independently by Kuhlet al (33). The induction does not require de novoprotein synthesis (6-8), and a nuclear factor(s) is present which specifically binds to hexamer motifs in both induced and uninduced cells (31); thus, intriguing possibility is that a given factor(s) can act both as a repressor (or silencer) in uninducedand as an activator in induced cells. This possibility needs to be investigated further by characterizing the nature of such a factor(s) in induced and uninduced cells. Recent studies demonstrate that other cytokine genes such as those of tumor necrosis factor (TNF) (36) and interleukin-6 (IL-6)--also as B cell stimulatory factor 2 (BSF-2), "IFN-fl2" or 26 kd protein (3739)--are induced by the same agents that induce IFN gene expression. Interestingly, sequences similar to the hexamer motifs within the IFN-fl gene are also found in the IFN-~, TNFs, and IL-6 genes, suggesting a possible commonrole for such sequences in the induction of these genes. However, the induction mechanisms of TNF and IL-6 genes by IFN inducers maydiffer from the induction mechanismsof those genes by other agents such as endotoxin in monocytes or mitogens in lymphocytes. It is particularly interesting that the IL-6 gene contains morethan two cap sites located significantly distant from each other (37). The use of different cap sites seemsto differ fromone cell type to the other, suggesting the presence

Annual Reviews CYTOKINE GENE REGULATION

445

of different control elementswithinthe genein different tissues. It also remains to be seen whether the induction of the IFN genes by other cytokines such as PDGF,IL-1, TNFs,and CSF-1is mediatedby the same sequencesresponsiblefor the virus-mediatedinduction of the genes.

Annu. Rev. Immunol. 1988.6:439-464. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

T Cell-Derived

Cytokine (Lymphokine) Genes

Following the activation of lymphocytes, expression of various lymphokinegenes is induced. Thecontrolled, coordinated expression of the lymphokine genes(as well as the lymphokine receptor genes)plays a crucial role in the proliferation, differentiation, and function of both T and B cells. In vivo, T cells are thoughtto be activated throughtheir recognition of nominalantigen on the surface of an antigen-presentingcell (APC) the context of major histocompatibilityantigens. This activation process involves the T cell antigen receptor/CD3complex(CD3/Ticomplex)whose triggering by antigen/MHC results in a cascade of biochemicalevents, such as membrane phosphatidylinositol turnover, calcium mobilization, and activation of protein kinase C (40). TheAPCalso providesyet another signal for T cell activation, IL-1, whoseintracellular signalling pathway remainsto be identified (41). In addition, various T cell antigens, CD2, CD28,and CD5appear to deliver activation signals (42-44). In vitro, cells are also activated by various agents including monoclonal antibodies against either the CD3or Ti, lectins such as phytohemagglutinin(PHA) or concanavalin-A (ConA), and 12-o-tetradecanoylphorbor-13-acetate (TPA)(40). However,none of these stimuli alone is able to induce phokine genes to maximallevels; a combinationof the various "signallings" delivered by those agents seemsto be required for optimalinduction of the lymphokine genes both in normalperipheral blood lymphocytes (PBLs)and in manyof the cultured T cell lines. Lectins and calcium ionophorescan substitute for the signal(s) normallyprovidedby antigen for the induction of lymphokine genes, while TPA,an activator of protein kinase C, can further increasethe inductionlevel (40). Most of the lymphokines described to date are produced by hel++ per/inducer T cells (CD4 T cells), but another T cell subset with a CD8 phenotypeis also capable of producing somelymphokines(45). In the mouse, the helper T cells have been classified in two categories, TH1 andTH2cells, originally basedon their difference in helper function for antibodyresponses(46) and subsequentlybasedon the difference in their pattern of lymphokineproduction (47). Studies by Mosmann et al showed that TH1-type T cell clones are capableof producinginterleukin-2(IL-2), interleukin-3 (IL-3), IFN-y, and granulocyte/macrophagecolony stimulating factor (GM-CSF),while TH2-typeT cell clones produce IL-3,

Annual Reviews

Annu. Rev. Immunol. 1988.6:439-464. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

446 TANIGUCHI interleukin-4 or B cell stimulatory factor-1 (IL-4 or BSF-1),and GM-CSF (47). Manyof the lymphokinegenes have been cloned, and their structure extensively analyzed. NormalT cells do not express detectable levels of lymphokinespecific mRNAs unless they are activated by mitogenicagents such as PHAand TPA.By applying the nuclear transcription assays on normal peripheral blood lymphocytes(PBLs), Kronkeet al demonstrated that induction of transcription of IL-2 and IFN-~genes as well as the proto-oncogene c-myc gene in humanPBLsdoes not require de novo protein synthesis, as the activation occursin the presenceof protein synthesis inhibitor, cycloheximide (48). Onthe other hand, Paetkauet al (49) reported that in T cell lines such as Jurkat and EL4.E1,cycloheximide appears to inhibit inducedIL-2 mRNA accumulationif it is present at the onset of induction, and this suggests a requirementfor protein synthesis in those cell lines. The difference betweenthe two groups maybe due to the use of different cell types or different control mechanisms.The expression level of a given lymphokineis also regulated by other lymphokines(or cytokines). In this regard, Grabsteinet al (50) foundthat mRNAs for IL-2 and IFN-7 show biphasic accumulations in mitogenactivated peripheral blood T cells. Theysuggested the involvementof IL-2 synthesis not in the early but in the late wave of IFN-~ mRNA accumulation. In addition to those studies, muchinformation now demonstratesthat various cytokinescan stimulate expressionof other cytokine genes (4, 5, 51). Whetheror not such cytokine-induced cytokine geneexpression involves the sameregulatory mechanisms operating in the mitogen-activated T cells is not clear at present. Usingcloned genes as probes, the effects of inhibitors of lymphokine geneexpression such as glucocorticoid hormonesand cyclosporin A(CsA) have been studied. Althoughthe inhibitory mechanismsof these agents are not yet known,the use of such inhibitors maygive someinsight into the differential regulatory mechanismsoperating in lymphokinegenes (52, 53). Whilethe well-characterized cases of glucocorticoid-mediated regulation of gene expressioninvolve the activation of target genes, the expression of most lymphokinegenes including IL-2, IFN-~, IL-3, and GM-CSF genes is suppressed by glucocorticoids such as dexamethasone (53). In contrast, another immunosuppressiveagent, CsA, appears inhibit selectively the inductionof certain lymphokine genes. Inhibition of the mRNA induction by CsAis observedin IL-2, IFN-~,, IL-3, and BSF1 (IL-4) genes, but not in GM-CSF genes (54-57). At least in the case the IL-2 gene, the inhibition ~vasshownto occurat the transcriptional level (54). Since GM-CSF is also producedby non T cells such as macrophages, endothelial cells, and fibroblasts (58, 59), a distinct mechanism may

Annual Reviews

Annu. Rev. Immunol. 1988.6:439-464. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

CYTOKINE GENE REGULATION

447

operative in the case of the GM-CSF gene. Comparisonof the putative regulatory DNA sequenceswithin the 5’-flanking sequences of manylymphokine genes revealed the presence of short stretches of homologies amongthe cloned lymphokine genes and LTRsequences of T lymphotrophicretroviruses (60-63). At present, however,little is known about the real function of such sequences. Cis-acting regulatory DNA sequences have been identified and characterized in the humanIL-2 gene. Sequenceanalysis of both humanand mouseIL-2 genes revealed the presence of unique, well-conserved DNA sequences within the Y-flanking region, ~uggestingthese sequencesmay have an important function in gene regulation (64). Fujita et al (63) localized functional DNAsequences required for humanIL-2 gene expressionin activated T cell lines. Thestrategy wasto excise possible regulatory DNAsequences from the IL-2 gene and fuse them to the chloramphenicolacetyltransferase (CAT)structural gene. The resulting fusion genes wereeach transfected into T cell and non-Tcell clones, and the transient expressionof the CATgene followingactivation by mitogens was monitoredby measuringCATactivity and/or by S 1 mappinganalysis of the transcripts. Theauthors foundthat sequencespresent within the 5’flanking region mediate mRNA transcription both in Con A-induced Jurkat and TPA-inducedEL-4cells. The Jurkat and EL-4 cells used in this experimentare sublines, whichrequire ConA and TPA,respectively, for the maximalinduction of the endogenousIL-2 gene (64). Induction specific transcription occurred in those T cell lines but not in non-T cells such as FL(fibroblast) andRaji (B lymphoblastoid)cells. A similar observationwasalso reported by Siebenlist et al, whoapplied essentially the sameexpressionsystem(65). Aseries of expressionstudies for the IL-2 generevealedthat: (a) the boundaryof the sequencesrequired for maximalinduction lines between positions -319 and -264 with respect to the IL-2 gene cap site both in Jurkat and EL-4cells; (b) a piece of DNA from -319 to - 127 functions in orientation independentmannerand activates heterologousIFN-fl promoter sequences in the induced T ceils; (c) sequences downstreamfrom -81 contain an inert promoter that is activated by a heterologous enhancer(i.e. the IFN-~inducibleenhancer)in non-Tcells, indicating that the promoterfunction per se is not restricted to T cells. Thefunctional, regulatory DNA sequencesof the humanIL-2 gene were further dissected into three core sequenceelements whichtogether confer maximalinducibility in mitogen-treated T cells (66, 67). Durand et al (68) also reported presenceof the inducible sequenceelementsthat showproperties similar to those described above, in a 275-bp DNAfragment spanning from -326 to -52 (i.e. XmnIsite--SspI site) of the humanIL-2 gene. This upstream

Annual Reviews

Annu. Rev. Immunol. 1988.6:439-464. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

448

TANIGUCHI

region apparently undergoesa conformational change in its chromatin structure after induction of the cells, becausethis region becomessusceptible to DNaseI digestion (65). Noevidencesuggests the presence negativeelementswithin this region. At present, the possibility cannotbe ruled out that other cis-acting regulatory sequencesmayalso be present in other regions of the geneandits surroundings. In the gibbon leukemiaT cell line, MLA144, the presence of the long terminal repeat (LTR)of the gibbon leukemia virus within the 3’ nontranslated region of the geneseemsto be responsiblefor the constitutive expressionof a rearrangedIL-2 gene but not the normalallelic IL-2 gene (69). This indicates that the basic promotersequenceof the IL-2 gene, reported by Fujita et al (63), can be activated by additional nearbyciselementswithout activating the 5’ regulatory sequences. Little is knownat present about the nature of nuclear factors that may interact with the regulatory elements described above. Recent data by Angelet al (70) demonstratedthat the nuclear factor AP-1binds to TPAresponsive elements (TREs)within the promoter regions of the genes for humanmetallothionein IIA and collagenase. The TREspossess a conserved 9 bp motif. Whencultured ceils are treated with TPA,the binding activity to the TREsincreases via a posttranslational mechanism. Angelet al also showedthat synthetic copies of the motif conferred TPA inducibility uponheterologous promotersand noted that a very similar motif is also present within the IL-2 gene(-185 to -176). In viewof the ¯ abovefindings and the fact that the IL-2 geneis inducibleby TPAin many T cell lines, the intriguing possibility is that AP-1is one of the factors involvedin IL-2 generegulation. It is interesting that a similar sequence motif is also found within the 5’-flanking region of the humanIFN-ygene (71). Attemptshave been madeto identify regulatory DNA sequencesfor the IFN-ygene whoseinduction is also regulated primarily at the level of transcription (48). In a waysimilar to the IL-2 gene, expressionof IFN-~ geneis also restricted to T cells. Hardyet al (72, 73) haveextensively analyzedthe conformationalalteration of the chromatinstructure of the humanIFN-~gene in various T and non-Tcell lines. ByperformingDNase I mappinganalysis, they have identified several DNaseI hypersensitive sites in cultured T cell lines such as Jurkat, HUT-78,and a subline of HUT-78 whichconstitutively expresses the gene. Thehypersensitive sites are localized either withinthe 5’-flankingregionor withinthe first intron of the gene. Whilesomeof the sites are also observedin non-Tcells, a prominentsensitive site is present approximately3.0 kb upstreamof the transcription initiation site only in T cells that are committed to express the IFN-~,gene. However,these authors mentionthat T cell lines trans-

Annual Reviews

Annu. Rev. Immunol. 1988.6:439-464. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

CYTOKINE GENE REGULATION

449

fected with CATgene constructs containing either 5’ or intron sequences (the precise sequences are not specified) failed to activate CATgene expression (73). On the other hand, Younget al (74) reported that stable transfection of a genomic 8.6 kb DNAfragment containing the human IFN-~ gene and its flanking sequences resulted in the induced expression of the gene in murine T cell lines but not in murine fibroblast cells, suggesting the presence of a regulatory element(s) within the DNAfragment. Although both IL-2 and IFN-~ genes can be induced in normal T cells with very similar kinetics of mRNA accumulation (50, 75), they may regulated by different mechanisms since: (a) The two genes seem to under different developmentalcontrol in T cells; neonatal T cells have an intrinsic deficiency in the expression of the IFN-~ gene but not the IL-2 gene (76). IL-2 gene expression is observed even in immature CD4-CD8"double-negative" thymocytes (77). (b) Expression of the IFN-~ gene induced by IL-2 (45, 50, 78). (c) Various T cell lines manifest different patterns of IL-2 and IFN-~ gene expression. For example, the IL-2 gene is inactive in T cells infected by humanT cell leukemiavirus type I (HTLV1) but not in T cells infected by the humanimmunodeficiencyvirus (HIV1), whereas the opposite situation is found in the case of the IFN-~gene (78). It is likely that multiple factors interact with the regulatory sequences of lymphokine genes which could in concert give rise to maximal expression. In other words, someof those factors mayinteract with various lymphokinegenes, while others maybe specific to a particular set of the genes. Amongthe hematopoietic growth factors, GM-CSFand IL-3 but not G-CSFand M-CSFare produced in normal T cells (58, 80). Like IL-2 and IFN-~genes, expression of the IL-.3 gene is observed almost exclusively in T cells, while the GM-CSF gene seems to be expressed also in nonlymphoid cells (58, 80). By comparing the sequences for the GM-CSFcDNAand chromosomalgenes, Stanley et al (62) provided evidence for the presence of distinct promoters within the mouseGM-CSF gene. The authors speculate on the differential control mechanismsfor those promoter sequences in the expression of the gene in different celt types. By applying the CAT assay system, Chan et al (81) provided evidence that a 660 bp segment within the Y-flanking region (upstream from the initiation ATG)of the humanGM-CSF gene contains a regulatory element(s) that functions an HTLV-l-infected T cell line following induction by mitogens. It is interesting that the hexanucleotide motif CCGCCC, which is found in promoter sequences of noncytokine genes such as SV40(82) and HSV-TK (83), is found upstream of the TATApromoter sequence of the mouse

Annual Reviews

Annu. Rev. Immunol. 1988.6:439-464. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

450

TANIGUCHI

GM-CSF and IL-3 genes (84). The hexanucleotide sequence within those SV40and TKpromoters specifically binds to a nuclear factor Spl and plays a crucial role in the expression of those genes (82, 83). Therefore, is possible that this sequencealso plays a role in sustaining transcription of those cytokine genes in concert with the conjectured, additional regulatory sequence elements of the gene. Little is knownat present about the sequence elements controlling the IL-3 gene in T cells. As shown in many other cytokine genes, the 5’flanking re#on of the IL-3 gene seems to show an area of markedsequence conservation between man and mouse (80), suggesting the importance these conserved regions in gene regulation. Miyatake et al (85) stably introduced a cloned mouse genomic DNAcontaining the IL-3 gene and its flanking region into mouseLtk- cells and detected constitutive production of IL-3 in the transfected L cells. Such unregulated expression of the gene may be due to a copy number effect, as the recipient cells each contained about a hundred copies of the transfected gene. The unique GC rich sequence (including the Spl site) present within the upstream region of the TATA sequence could also be responsible partly for the derepressed expression of the gene. In addition to T cells, B cells also appear to produce some cytokines (86-88) whose regulation is just beginning to be explored. Other

Cytokine

Genes

At present, little information is available about the transcriptional regulation of most other cytokine genes. IL-1, originally described as a product of activated macrophages, is produced by a variety of cells, including endothelial cells, lymphocytes, and epidermal.cells (89, 90). Production of IL-1 can be induced in vitro by various stimuli such as bacterial endotoxins, silica, TPA, IFN-~, and TNF. TwoIL-1 species, IL-I~ and IL-lfl, with very similar biological activities were identified, and their genes have been cloned (92-94). The genes are structurally related to each other, but their expression seems to be regulated independently. For example, Acres et al (95) observed that humanT cell clones, IL--I~ .mRNAaccumulates predominantly following stimulation of the cells by combinations of anti-CD3, IL-2, and TPA. On the other hand, in both human peripheral blood monocytes and a monocytic leukemia cell-line, IL-lfl mRNA accumulation is predominant in cells stimulated by lipopolysaccharide (LPS). While the IL-I~ mRNA could not be detected, a low constitutive level of IL-lfl mRNA expression was observed in unstimulated human peripheral blood macrophages (93). Expression of the genes for two species of TNFalso seems to be regulated by independent mechanisms. TNF-~ and TNF-fl genes are struc-

Annual Reviews CYTOKINE GENEREGULATION 451 turally related, and they are separated by a spacer sequence of only 795 bp (96-98). Expression of the TNF-~ and TNF-fl genes is induced activated monocytes and in T cells, respectively. Accordingly, the 5’flanking regions of both genes show few sequence homologies to each other (95, 96). It is likely that sequence elements that respond to the cellular activation signals differ from those involved in the virus-induced expression of the genes (36).

Annu. Rev. Immunol. 1988.6:439-464. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

General Remarks In general, it is important to bear in mind that gene expression can be controlled at manylevels, as is outlined below. Accumulation of mRNA often may not be due to the transcriptional activation of the gene. For example, evidence for the effect of TPAas well as cycloheximide in the modulation of a specific mRNA degradation pathway has been provided (13, 26, 59, 99-101). Thus, ongoingtranscription does not always correlate with the accumulation of mRNA transcripts (13, 59). Muchof the work cited here therefore needs to be interpreted carefully in building models for the regulation of transcription. At present, little is knownabout howthe induced transcription of genes is shut off. A plausible idea is that nuclear factors that regulate the transcription becomeactive only transiently after cellular induction through specific modifications such as phosphorylation or allosteric changes in the factors, and soon afterward they go back to their steady state forms.

POSTTRANSCRIPTIONAL CYTOKINE GENES

REGULATION OF

Oneof the characteristic features of the cytokine genes is that most of the genes are transiently expressed. This transient nature in gene expression is supposed to be an important aspect of the control of the proliferation, differentiation, and function of cells, since overproductionof the cytokines mayresult under certain circumstances in the dysregulation or dysfunction of cells (see below). Hence, the expression of the genes are controlled not only at the transcriptional level, but at the posttranscriptional level. Particularly notable is the fact that the cytokine mRNAs have a relatively short half-life compared to mRNA such as fl-globin mRNAwhich has a half-life of greater than 17 hr. For example, the half-life of the GM-CSF mRNA in PHA-inducedT cells is less than 30 min (101). In the case IFN-fl and IL-2 mRNA,the half-lives seems to be less than 2 hr in poly(rI):poly(rC)-induced human fibroblasts and in the TPA-induced mouseT cell line EL-4, respectively (13, 20, 63). Thus, in addition to the temporal induction of mRNA transcription, selective mRNA degradation

Annual Reviews

Annu. Rev. Immunol. 1988.6:439-464. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

452 TANIGUCHI also plays a crucial role in controlling the level and duration of cytokine production. Raji & Pitha (13) have shownthat the induction of the IFN-fl gene by poly(rI) : poly(rC)results in the activation of transcription, but stabilization of the mRNA after induction contributes to the maintenance of mRNA levels. In addition, the importanceof the mRNA stabilization was emphasizedby Nir et al (26): they showedthat IFN-fl mRNA transcription of the humangene introduced in mousecells occurred at about the samerate whetheror not the cells wereinducedby poly(rI) : poly(rC) or cycloheximidebut that the mRNA accumulation occurred in induced but not in uninducedcells. Their results can be best explained by the stabilization of the mRNA in the induced cells. In view of the welldocumentedfact that cycloheximide causes accumulation of IFN-fl mRNA, a plausible model is that the mRNA accumulation is due to blockageof de novosynthesis of a short-lived protein(s) that is induced in the cells and plays a role in the selective control of the certain mRNA levels. Such a modelhas been evokedby Efrat & Kaempferfor IL-2 gene regulation (99): Theyshowedthat the shut-off of the induced IL-2 mRNA accumulationis circumventedby cycloheximide.Onthe other hand, Thorens et al (59) have shown that induction of the GM-CSF mRNA macrophages by LPSis not the result of induction or enhancement of gene transcription but the result of mRNA stabilization and that induction of mRNA dependson ongoing protein synthesis. Their results suggest that LPSinduces the synthesis of a protein(s) that prevents mRNA degradation. Common sequences that appear to confer instability on the cytokine mRNAs have been proposedand experimentally verified, at least in the case of GM-CSF mRNA. Twogroups observed that particular motifs of AU-rich sequences are present commonlywithin the 3’ nontranslated regions of mRNA encodingmanyof the cytokines as well as someof the proto-oncogenes(101,102). Significantly, the AUrich regions weremore highly conserved than the protein coding regions between humanand mouseGM-CSF and TNF(102). In order to test whether or not the GM-CSF mRNA instability is due to such AUrich sequences in the 3’ nontranslatedregion, Shaw&Kamen (101) first inserted a synthetic 51 DNA containing the ATrich sequences into the 3’ noncodingregion of the rabbit fl-globin gene. Byperforminga series of expressionstudies, the authors showedthat DNA with ATrich sequences, but not control DNA with the samelength, rendered the otherwise very stable globin mRNA as unstable as the GM-CSF mRNA. It remains to be experimentally tested whether or not all the mRNAs containing the commonsequence motifs such as A-U-U-U-Aor U-U-A-U-U-U-A-U are responsible for the

Annual Reviews

Annu. Rev. Immunol. 1988.6:439-464. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

CYTOKINE GENE REGULATION

453

mRNA instability; however,this is likely in viewof the results of Shaw& Kamen, together with the fact that mostof the genescontainingthe motifs are cytokinegenes and proto-oncogeneswhoseexpression is inducedtransiently by extracellular stimuli (101, 102). As with studies on IFN-fl and IL-2 tnRNAs,Shaw& Kamenalso provided evidence for the involvement of de novoprotein synthesis in the mRNA stabilization by showingthat cycloheximide stabilized the "unstable" globin mRNA with the AU sequences.Hereagain, an intriguing possibility is that the presenceof an unstable protein factor(s) maycontribute to the posttranscriptional control of the cytokinegenes. Regulationof the cytokinegeneexpressioncould occur also at the level of mRNA processing. Evidencefor the production of distinct forms of cytokines by alternative splicing of mRNA precursors has been provided for G-CSF(103) and M-CSF(80, 104, 105). However,the biological significanceof the differential splicing in these twogenesis not clear at present. DYSREGULATION

OF CYTOKINE

GENES

Cytokinesplay a crucial role in transmitting signals for proliferation, differentiation, and function of various target cells, so it is likely that dysregulation of cytokine genes and their receptors could result in or contribute to the generationof various diseases. In fact, several examples supporting such a notion have accumulated. Disordered expression of various cytokines in rheumatoidarthritis has been documented in a number of studies (106-108).Constitutive productionof IFN-~,a suppressive factor for hematopoesis,maybe involved in the aplastic anemia(109). Leunget al (110) report the possible involvementof IL-1 and IFN Kawasakisyndrome,as the two cytokines render cultured vascular endothelial cells susceptibleto lysis by antibodiespresentin the circulation of patients. Muchattention has been focusedon the dysregulationof cytokinegenes and their receptor genesin malignanttransformationof cells. In general, aberrant expression of various growthfactors and receptors appears to play a major role in manyof the cellular transformationprocesses, most likely as the consequence of the dysregulationand/or structural alteration of those genes (for general review, see 111, 112). I focus belowon the dysregulationof cytokinegenes in leukemogenesis. Involvementof the IL-2 system in T cell neoplasmshas been studied particularly in relation to the development of adult T cell leukemia/lymphoma (ATL). Most if not all of ATLcells are mature, peripheral CD4÷ CD8 - (i. e. IL-2producingcells) T cells (113)andare thought

Annual Reviews

Annu. Rev. Immunol. 1988.6:439-464. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

454 TANIGUCHI to be caused by a lymphotropicretrovirus, HTLV-1 (114-116). ATLcells as well as T cell lines transformedin vitro by HTLV-1 display constitutive expressionof receptorsfor IL-2 (IL-2R;Tac-antigen)on their cell surface, and some of them apparently produce IL-2 spontaneously (117-119). Analysis of the provirus genomeof HTLV-1revealed the absence of a typical oncogenebut the presence of a region encodinga unique nuclear protein, tat I (or p40X),that functionsas tr ans-activator(120-122). In view of these findings, a plausible idea for the initial step of the leukemogenesis wasthat the trans-activator protein functions also to induce aberrant expression of the IL-2R and IL-2 genes. Thus, a series of expression studies have been carried out, focusing on the activation of transcription for the genes encoding IL-2 and IL-2Rmediated by tat I (p40x) and/or mitogenicsignals. Maruyama et al (123) showedthat distinct DNA sequencesin the Y-flankingregion of both the IL-2 and IL-2Rgenes wereresponsiveto transcriptional activation by tat I (p40x) in Jurkat cells but not in other non-Tcells tested. Activation by tat I is particularly pronouncedfor the IL-2R gene but not for the IL-2 gene (123, 124). Similar experimentswere carried out with the IL-2Rgene by Cross et al (125), and they providedevidence for the trans-activation of the IL-2R gene sequencesin Jurkat and an HTLV-1-infected T cell line MT-2.Inoue et al (126) showedthe induction of IL-2RmRNA (and marginal induction of IL-2 mRNA) in Jurkat cells transfected with an expression plasmidfor tat I. Thoseresults suggest a mechanism by whichHTLV1-infected T cells mayescapethe constraints of normalcellular growth;efficient inductionof the IL-2Rby tat I in infected T cells maysustain cell growthwithoutany further antigenic stimuli (perhapsby a paracrine mechanism, since the IL2 geneis not efficiently activatedby the trans-activatoralone). Significantly, IL-2 genesequencesare synergistically activated by the combinationof tat I (p40x) expressionandsubsequentextracellular stimulation by concanavalin-Aor anti-CD3(123, 124). Takentogether, one mayenvisage that the chancefor growthautonomyof a given T cell clone +, IL-2 producer T cell) increases greatly in vivo when (presumably,CD4 it is (a) infected by HTLV-1 and (b) exposedcontinuouslyto a specific antigensuch as viral antigen or infectious agents, since this wouldlead to aberrant activation of the IL-2 autocrine loop (two step activation model; Figure 2; 123). Suchan event might be crucial in developingATLat an early stage. Operation of the aberrant IL-2 autocrine stimulation may render the T cell proneto developthe malignantstate of the cell, wherein the IL-2 systemis not necessarilyoperativeanymore.In fact, in mostcases of ATL,the leukemicT cells do not produceIL-2 and respond poorly to IL-2 in vitro, suggestingthe occurrenceof an additional event(s) in the establishment of ATLin vivo (127). On the other hand, someATLcells

Annu. Rev. Immunol. 1988.6:439-464. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

Annual Reviews CYTOKINE GENE REGULATION 455

Annual Reviews

Annu. Rev. Immunol. 1988.6:439-464. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

456

TANIGUCHI

manifest IL-2 dependencyfor their in vitro growth (128). In addition, IL2 autocrine stimulation seems to account directly for the in vitro proliferation of someATLcells (129). It remains to be seen howsuch in vitro properties of ATLcells correlate with their leukemogenicgrowth in vivo. To examine whether IL-2 autocrine growth stimulation contributes to the generation of tumorigenic T cells, Yamadaet al (130) constructed retrovirus whichuponinfection of cultured cells gives rise to the production of humanIL-2. Whenan IL-2-dependent murine T cell line, CTLL-2,was infected with the virus, the cells gained growth autonomyin vitro under certain conditions and developed tumors (lymphomas) when injected into nude and syngeneic mice. This result suggests further the importance of the aberrant operation of the IL-2 autocrine loop in the development and/or maintenance of T lymphoma/leukemia. The involvement of the IL2 autocrine mechanismin T cell malignancy has been also reported in nonHodgkinT cell lymphoma(13 l) and gibbon T cell leukemia (69). In B cells, evidence has been provided for the involvementof the Epstein Barr virus-induced autocrine growth stimulation by a B cell growth factor in the process of cellular transformation (112, 132, 133); this suggests the role of the autocrine stimulation in the virus-induced B lymphoma development. Dysregulation of hematopoetic growth factor genes also seems to be involved in leukemogenesis. Except for the G-CSFgene, genes for GMCSF, IL-3, M-CSF,and the M-CSFreceptor (i.e. c-fms), as well as the genes for other growth factors and growth factor receptors appear to be clustered on the long arm of human chromosome 5. Deletions in the long arm of this chromosomeare frequently observed in patients with myelodysplastic syndrome and acute nonlymphocytic leukemia (80, 134). The relationship between the chromosomal deletions and myeloid disorders needs to be further investigated. During the process of myeloblastic leukemogenesis that is induced by Friend leukemia virus in mouse, abnormal responsiveness to GM-CSF of the infected cells seems to be crucial in developing the cell growth autonomyprior to cell transformation (135). In the mouse myelomonocytic leukemia line WEHI-3B,Ymeret al (136) have shownthat the constitutive expression of IL-3 is due to a retroviral insertion in the promoter upstream region of the gene, suggesting the possible involvementof autocrine stimulation by this growth factor in the early phase of the leukemogenesis. A role for dysregulated autocrine growth stimulation in malignant transformation of hemopoiefic cells has be~n clearly demonstrated by Lang et al (137). They introduced a murine GM-CSF gene in a factor dependent murine cell line (FDC-P1), and this resulted not only in autonomousgrowth in vitro but also converted the cells into a tumorigenic phenotype. In chicken hematopoietic cells, several

Annual Reviews CYTOKINE GENE REGULATION

457

oncogenesof the src family as well as v-rnil appear to abrogate dependency on exogenous growth factors by inducing dysregulated expression of growth factor genes (138, 139).

Annu. Rev. Immunol. 1988.6:439-464. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

SUMMARY Following the isolation and characterization of manycytokine genes, we began to understand the mechanismsregulating cytokine gene expression. Needless to say, understanding the mechanisms by which induction of gene expression occurs in a transient, cell type-specific mannerin response to extracellular inducers is a central issue in eukaryotic molecularbiology. The cytokine systems represent suitable models for studying the mechanisms regulating gene expression, since the expression of cytokines appears to be tightly regulated by restricted types of cells and inducers. At present, cis-acting DNAsequences involved in gene regulation have been identified in only a few cytokine genes. Little is knownabout the nature of factors regulating the cytokine gene expression. Of particular interest are the mechanismsby which the extracellular stimulation of the cells delivers signals in the nucleus and howthey turn on the otherwise nonoperative transcription machinery. In manycases, the induction of the genes will involve the activation of preexisting factors rather than the de novo protein synthesis by modifications of the factors. Dysregulation of cytokine gene expression may be caused by chromosomalalterations or by infection of viruses that induce activation or inactivation of the expression machinery. This process maybe crucial in the etiology of various diseases including neoplasms. To understand the complexnature of cytokine action in the regulation of cell proliferation, differentiation, and function, more attention should also be focused on the genes encoding the respective receptors. ACKNOWLEDGMENTS

I thank Drs. C. Weissmann, T. Maniatis, and T. Kishimoto for sharing unpublished data and discussion. I also thank Drs. E. Clark, E. Barsoumian, T. Fujita, G. Yamada, M. Hatakeyama, and H. Harada for valuable advice and comments, and Ms. M. Sonoda-Nagatsuka for excellent assistance. Literature Cited 1. Dumonde,D. C., Wolstencroft, R. A., Panayi, G. S., Matthew,M., Morley, J., Howson, W. T. 1969. "Lymphokines": Non-antibody mediators of cellular

immunity generated by lymphocyte activation. Nature 224:38 2. Cohen, S. 1977. The role of cellmediated immunity in the induction of

Annual Reviews

Annu. Rev. Immunol. 1988.6:439-464. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

458

TANIGUCHI

inflammatoryresponses. Am.J. Pathol. 88(3): 502 3. Merrill, J. E. 1987.Macroglia:neural cells responsive to lymphokinesand growthfactors. Immunol.Today8(5): 146 4. Farrar, J. J., Benjamin, W.R., Hilfiker, M.L., Howard,M., Farrar, W.L., Fuller-Farrar, J. 1982. Thebiochemistry, biologyandrole of interleukin 2 in the inductionof cytotoxic T cell and antibody-formingB cell responses. Immunol. Rev. 63:129 5. Kohase, M., May, L. T., Tamm,I., Vilcek,J., Slehgal, P. B. 1987.A cytokine network in humandiploid fibroblasts: interactionsof beta interferons, tumornecrosis factor, platelet-derived growthfactor and interleukin-1. Mol. Cell. Biol. 7:273 6. Stewart, W.E. II. 1981.The Interferon System. NewYork: Springer-Verlag. 2nd ed. 7. Lengyel,P. 1982. Biochemistryofinterferons andtheir actions. Ann.Rev.Biochem.51: 251 8. Weissmann,C., Weber, H. 1986. The interferon genes.Prog.Nucl. AcidsRes. 33:251 9. Zullo, J. N., Cochran,B. H., Huang,A. S., Stiles, C. D. 1985.Platelet-derived growth factor and double-stranded rlbonuclelc acids stimulate expression of the samegenesin 3T3cells. Cell 43: 793 10. Moore,R. N., Larsen, H. S., Horohov, D. W., Rouse, B. T. 1984. Endogenous regulation of macrophage proliferative expansionby colony-stimulation-factor-induced interferon. Science 223: 178 11. Warren,M. K., Ralph, P. 1986. Macrophage growth factor CSF-1stimulates humanmonocyteproduction of interferon, tumornecrosis factor, and colony stimulating activity. J. Immunol. 137:2281 12. Van Damme, J., DeLey, M., Opdenakker,G., Billiau, A., DeSomer, P., Van Beeumen, J. 1985. Homogeneous interferon-inducing 22K factor is related to endogenouspyrogen and interleukin-1. Nature 314: 266 13. Raji, N. B. K., Pitha, P. M. 1983.Two levels of regulationof fl-interferongene expressionin humancells. Proc. Natl. Acad. Sci. USA80:3923 14. Weidle, U., Weissmann,C. 1983. The Y-flanking region of a humanIFN-ct gene mediatesviral induction of transcription. Nature303:442 15. Ohno,S., Taniguchi,T. 1983. The5’-

flanking sequenceof humaninterferonfi geneis responsiblefor viral induction of transcription. Nucl. AcidsRes. 11: 5403 16. Ohno,S., Taniguchi,T. 1981.Structure of a chromosomal gene for human interferon ft. Proc. Natl. Acad.Sci. USA78:5305 17. Ragg, H., Weissmann,C. 1983. Not morethan 117 base pairs of Y-flanking sequence are required for inducible expression of a humanIFN-ct gene. Nature 303:439 18. Ryals, J., Dierks, P., Ragg, H., Weissmann,C. 1985. A 46-nucleotide promoter segmentfrom an IFN-a gene renders an unrelated promoter inducible by virus. Cell 41:497 19. Hiscott, J., Cantell, K., Weissmann, C. 1984.Differential expressionof human interferon genes. Nucl. Acids Res. 12(9): 3727 20. Pitha, P. M., Ciufo, D. M., Kellum, M., Raji, N. B. K., Reyes, G. R., Hayword,G. S. 1982. Induction of humanfl-interferon synthesis with poly(rI-rC) in mousecells transfected with cloned cDNAplasmids. Proc. Natl. Acad. Sci. USA79:4337 21. Tavernier, J., Gheysen,D., Duerinck, F., Vander Heyden,J., Fiers, W.1983. Deletion mappingof the inducible promoter of humanIFN-fl gene. Nature 301:634 22. Maroteaux,L., Kahara, C., Mory,Y., Groner, Y., Revel, M. 1983. Sequences involvedin the regulated expressionof the humaninterferon-fl gene in recombinant SV40DNAvectors replicating in monkeyCOScells. EMBO J. 2:325 23. Zinn, K., DiMaio, D., Maniatis, T. 1983. Identification of two distinct regulatory regions adjacent to the humanfl-interferon gene. Cell 34:865 24. Fujita, T., Ohno,S., Yasumitsu,H., Taniguchi, T. 1985. Delimitation and properties of DNAsequencesrequired for the regulated expression of human interferon-fl gene.Cell41:489 25. Dinter, H., Hauser, H., Mayr, U., Lammers,R., Bruns, W., Gross, G., Collins, J. 1983. Humaninterferonbeta and co-inducedgenes: molecular studies. In TheBiologyof the Interferon System 1983, ed. E. De Maeyer, H. Schellekens, p. 33. Amsterdam: ElsevierSci. 26. Nir, U., Cohen,B., Chen,L., Revel,M. 1984. A humanIFN-fll gene deleted of promotersequencesupstreamfrom the TATA box is controlled post-transcriptionally by dsRNA.Nucl. Acids Res. 12:6979

Annual Reviews

Annu. Rev. Immunol. 1988.6:439-464. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

CYTOKINEGENE REGULATION 27. Fujita, T., Shibuya, H., Hotta, H., Yamanishi, K., Taniguchi, T. 1987. Interferon-fl generegulation: tandemly repeated sequencesof a synthetic 6 bp oligomerfunction as a virus-inducible enhancer. Cell 49:357 28. Fujita, T., Taniguchi,T. 1987. Unpublished data 29. Goodbourn,S., Zinn, K., Maniatis, T. 1985. Human t-interferon gene expressionis regulated by an inducible enhancerelement.Cell 41: 509 30. Maniatis, T. 1987. Personal communication 31. Fujita, T., Sakakibara,J., Sudo,Y., Kimura, Y., Taniguchi, T. 1987. Unpublisheddata 32. Dinter, H., Hauser, H. 1987. Cooperative interaction of multiple DNA elements in the humaninterferon-/~ promoter.Eur. J. Biochem.166:103 33. Kuhl,D., de la Fuente,J., Chaturvedi, M., Parimoo,S., Ryals, J., Mayer,F., Weissmann, C. 1987. Reversiblesilencing of enhancersby sequencesderived from the humanIFN-~tpromoter.Cell. In press S., Burstein, H., Maniatis, 34. Goodbourn, T. 1986. Thehumant-interferon gene enhanceris undernegativecontrol. Cell 45:601 35. Zinn, K., Maniatis,T. 1986.Detection of factors that interact with the human t-interferon regulatory region in vivo by DNAase footprinting. Cell45:611 36. Wong,G., Goeddel, D. 1986. Tumor necrosis factors ~ andfl inhibit virus replication and synergize with interferons. Nature323:819 37. Weissenbach,J., Chernajovsky, Y., Zeevi,M., Shulman,L., Soreq,H., Nit, U., Wallach,D., Perricandet,M., Tiollais, P., Revel,M.1980.Twointerferon mRNAs in humanflbroblasts: in vitro translation andEscherichiacoli cloning studies. Proc.Natl. Acad.Sci. USA77: 7152 38. Content, J., De Wit, L., Pierard, D., Derynk,R., DeClercq, E., Fiefs, W, 1982. Secretory proteins induced in humanfibroblasts under conditions usedfor the productionof interferonft, Proc. Natl. Acad. Sci. USA79:2768 39. Yasukawa,K., Hirano, T., Watanabe, Y., Muratani, K., Matsuda,T., Nakai, S., Kishimoto,T. 1987. Structure and expressionof humanB cell stimulatory factor-2 (BSF-2/IL-6)gene. EMBO J. In press Weiss, A., Imboden, J., Hardy, K., 40. Manger,B., Terhorst, C., Stobo, J, 1986.Therole of the T3/antigenreceptor complexin T-cell activation. Ann.

459

Rev. Immunol.4:593 41. Durum, S. K., Schmidt, J. A., Oppenheim, J. J. 1985. Interleukin 1: An immunologicalperspective. Ann. Rev. Irnmunol.3:263 42. Meuer,S. C., Hussey, R. E., Fabbi, M., Fox, A., Acuto,O., Fitzgerald, A., Hogdon,J. C., Protentis, J. P., Schlossman,S. F., Reinherz, E. L. 1984. Analternative pathwayof T cell activation: a functional role for the 50 kD T11sheeperythrocytereceptor protein. Cell 36:897 43. Martin, P. J., Ledbetter, J. A., Morishita, Y., June, C. H., Beatty, P. G., Hansen,J. A. 1986.A44 kilodaltoncell surface homodimerregulates interleukin 2 production by activated human T lymphocytes. J. Immunol. 136(9): 3282 44. Ledbetter, J. A., Parsons,M., Martin, P. J., Hansen,J. A., Rabinovitch, P. S., June, C. H. 1986. Antibodybinding to CD5(Tp67) and Tp44.Tcell surface molecules:effects on cyclic nucleotides, cytoplasmic free calcium, and cAMPmediated suppression. J. lmmunol. 137(10):3299 45. Kasahara,T., Hooks,J. J., Dougherty, S. F., Oppenheim, J. J. 1983. Interleukin 2-mediated immuneinterferon (IFN-y) production by humanT cells and T cell subsets. J. Immunol.130(4): 1784 46. Tada, T., Takemori,T., Okumura,K., Nonaka, M., Tokuhisa, T. 1978. Two distinct typesof helperT cells involved in the secondary antibody response: independentand synergistic effects of Ia- andIa+ helper T cells. J. Exp.Med. 147:446 47. Mosmann,T. R., Cherwinski, H., Bond,M. W., Giedlin, M. A., Coffman, R. L. 1986. Twotypes ofmurinehelper T cell clone I. Definitionaccordingto profiles of lymphokineactivities and secreted protein. J. Immunol.136(7): 2348 48. Kronke, M., Leonard, W. J., Depper, J. M., Greene,W.C. 1985. Sequential expression of genes involved in human T lymphocyte growth and differentiation. J. Exp. Med.161:1593 49. Paetkau,V., Shaw,J., Elliott, J. F., Havele,C., Pohajdak,B., Bleackley,R. C. 1987. Expressionpatterns of lymphokines and related genes. In Lymphokines, ed. D. R. Webb,D. Goeddel, 13: 73. NewYork: Academic 50. Grabstein, K., Dower,S., Gillis, S., Urdal, D., Larsen, A. 1986. Expression of interleukin-2, interferon-y, andthe IL2 receptor by humanperipheral

Annual Reviews

Annu. Rev. Immunol. 1988.6:439-464. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

460 TANIGUCHI bloodlymphocytes.J. Immunol.136(12): 4503 51. Oppenheim, J. J., Luger,T., Sztein, M. B., Steeg,P. S. 1982.Circuitofcytokine cell interactions that regulate immunological and inflammatory reaction. In Role of Macrophages in SelfDefence Mechanisms,ed. Z. Cohn,N. Ishida, p. 127. Tokyo:Tokyo Univ. Press 52. Shevach, E. M. 1985. The effects of cyclosporin A on the immunesystem. Ann. Rev. Immunol.3:397 53. Culpepper, J., Lee, F. 1987. Glucocorticoid regulation of lymphokine production by murine T lymphocytes. See Ref. 49, 13:275 54. Kronke, M. W., Leonard, W. J., Depper, J. M., Arya, S. K., WongStaal, F., Gallo, R. C., Waldmann, T. A., Greene, W.C. 1984. Cyclosporin A inhibits T cell growthfactor gene expression at the level of mRNA transcription. Proc. Natl. Acad. Sci. USA 81:5214 55. Granelli-Piperno,A., Inaba, K., Steinman,R. M. 1984. Stimulation of lymphokinerelease from T lymphoblasts: Requirementfor mRNA synthesis and inhibition by cyclosporin A. J. Exp. Med. 160:1792 56. Elliott, J. F., Lin,Y., Mizel,S. B., Bleackley, R. C., Harnish,D. G., Paetkau, V. 1984. Induction of interleukin 2 messenger RNAinhibited by cyclosporin A. Science 226:1439 57. Bickel, M., Tsuda, H., Amstad, P., Evequoz, V., Mergenhagen, S. E., Wahl, S. M., Pluznik, D. H. 1987. Differential regulation of colonystimulating factors and interleukin 2 production by cyclosporin A. Proc. Natl. Acad. Sci. USA84:3274 58. Metcalf, D. 1984. The Hemopoietic ColonyStimulating Factors. Amsterdam:Elsevier Sci. 59. Thorens,B., Mermod, J.-J., Vassalli, P. 1987. Phagocytosis and inflammatory stimuli induce GM-CSF mRNA in macrophages through posttranscriptional regulation. Cell 48:671 60. Fujita, T., Takaoka,C., Matsui, H., Taniguchi, T. 1983. Structure of the humaninterleukin-2 gene. Proc. Natl. Acad. Sci. USA80:7437 61. Holbrook,N. J., Lieber, M., Crabtree, G. R. 1984. DNAsequence of the 5’ flanking region of the humaninterleukin 2 gene: homologieswith adult T-cell leukemiavirus. Nucl. AcidsRes. 12(12): 5005 62. Stanley, E., Metcalf, D., Sobiezczuk, P., Gough,N. M., Dunn,A. R, 1985.

The structure and expression of the murine gene encoding granulocyte-macrophagecolony stimulating factor: evidencefor utilisation of alternative promoters. EMBO J. 4(10): 2569 63. Fujita, T., Shibuya, H., Ohashi, T., Yamanishi, K., Taniguchi, T. 1986. Regulation of humaninterleukin-2 gene: functional DNAsequences in the 5’ flanking region for the gene expressionin activated T lymphocytes. Cell 46:401 64. Fuse, A., Fujita, T., Yasumitsu,H., Kashima, N., Hasegawa, K., Taniguchi, T. 1984.Organizationand structure of the mouseinterleukin-2 gene. Nucl. Acids Res. 12:9323 65. Siebenlist, U., Durand,D.B., Bressler, P., Holbrook, N. J., Norris, C. A., Kamoun,M., Kant, J. A., Crabtree, G. R. 1986. Promoter region of interleukin-2 gene undergoes chromatin structure changesand confers inducibility on ehloramphenicol acetyltransferase geneduringactivation of T cells. Mol.Cell. Biol. 6:3042 66. Taniguchi,T., Fujita, T., Hatakeyama, M., Mori, H., Matsui, H., Sato, T., Hamuro,J., Minamoto,S., Yamada, G., Shibuya, H, 1986. Intefleukin-2 and its receptor: structure and functional expression of the genes. CoM Spring HarborSymp.Quant.Biol. LI: 577 67. Shibuya, H., Taniguchi, T. 1987. Unpublisheddata 68. Durand, D. B., Bush, M. R., Morgan, J. G., Weiss,A., Crabtree, G. R. 1987. A 275 basepair fragmentat the 5’ end of the interleukin 2 gene enhances expression from a heterologous promoterin responseto signals fromthe T cell antigenreceptor. J. Exp.Med.165: 395 69. Chen,S. J., Holbrook,N. J,, Mitchell, K. F., Vallone,C. A., Greengard,J. S., Crabtree, G. R., Lin, Y. 1985. A viral long terminalrepeat in the interleukin 2 geneof a cell line that constitutively produces interleukin 2. Proc. Natl. Acad. Sci. USA82:7284 70. Angel, P., Imagawa, M., Chiu, R., Stein, B., Imbra,R. J., Rahmsdorf,H. J., Jonat, C., Herrlich, P., Karin, M. 1987. Phorbol ester-inducible genes contain a commoncis element recognized by a TPA-modulated trans-acting factor. Cell 49:729 71. Gray, P. W. Goeddel, D. V. 1982. Structure of the humanimmuneinterferon gene. Nature 298:859 72. Hardy,K. J., Peterlin, B. M., Atchison,

Annual Reviews

Annu. Rev. Immunol. 1988.6:439-464. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

CYTOKINEGENE REGULATION R. E., Stobo, J. D. 1985.Regulationof expression of the humaninterferon-), gene. Proc. Natl. Acad.Sci. USA61: 542 73. Hardy,K. J., Manger,B., Newton,M., Stobo, J. D. 1987. Molecularevents involved in regulating humaninterferon-7 gene expression during T cell activation. J. ImmunoL 7:2353 74. Young,H. A., Dray, J. F., Farrar, W. L. 1986. Expression of transfected humaninterferon-~, DNA:evidencefor cell specificregulation.J. Immunol. 136: 4700 75. Efrat, S., Pilo, S., Kaempfer, R. 1982. Kinetics of induction and molecular size of mRNAs encodinghumaninterleukin-2 and ~-interferon. Nature297: 236 76. Lewis,D. B., Larsen,A., Wilson,C. B. 1986. Reduced interferon-gamma mRNA levels in humanneonates: evidencefor an intrinsic T cell deficiency independentof other genes involvedin T cell activation. J. Exp. Med.163: 1018 77. McGuire,K. L., Rothenberg, E. V. 1987. Inducibility of interleukin-2 RNAexpression in individual mature and immature T lymphocytes. EMBO J. 6(4): 939 78. Pearlstein, K. T., Palladino, M. A., Welte, K., Vilcek, J. 1983. Purified humaninterleukin-2 enhancesinduction of immune interferon. Cell. Immunol. 80: I 79. Arya, S. K., Gallo, R. C. 1987.T cell growth factor (Intedeukin 2) gene: Organizationand expression in human T lymphotropicvirus-infected cells in leukemia and AIDS.See Ref. 49, 13: 35 80. Clark, S, C., Kamen,R. 1987. The humanhematopoietic colony stimulation factors. Science236:1229 81. Chan, J. Y., Slamon,D. J., Nimer,S. D., Golde, D. W., Gasson,J. C. 1986. Regulation of expression of human granulocyte/macrophage colony-stimulation factor. Proc. NatLAead.Sci. USA 83:8669 82. Gidoni, D., Kadonaga,J. T., BarreraSaldana, H., Takahashi, K., Chambon, P., Tjian, R. 1985.Bidirectional SV40 transcription mediatedby tandemSpl bindinginteractions. Science230:511 83. McKnight,S. L., Kingsbury, R. C,, Spence,A., Smith,M.1984. Thedistal transcription signals of the herpes virus tk gene share commonhexanucleotidecontrol sequences.Cell 37: 253 84. Miyatake,S., Otsuka,T., Yokota,T,,

461

Lee,F., Arai, K. 1985.Structureof the chromosomalgene for granulocytemacrophage colonystimulating factor: comparison of the mouse and human genes. EMBO J. 4(10): 2561 85. Miyatake,S., Yokota,T., Lee, F., Arai, K. 1985. Structure of the chromosomal gene for murineinterleukin 3. Proc. Natl. Acad. Sci. USA82:316 86. Yoshizaki, K., Kanagawa,T., Fukunaga, K., Tseng, L. T., Yamamura, Y., Kishimoto, T. 1984. Isolation and characterization of B cell differentiation factor (BCDF) secreted from a humanB lymphoblastoid cell lines. J. Immunol. 132:2948 87. Jurgensen, C. H., Ambrus,J. L. Jr., Fauci, A. S. 1986.Productionof B cell growth factor136:4542 by normalhumanBcells. J. Immunol. 88. Wakasugi,H., Rimsky,L., Mahe,Y., Kamel,A. M., Fradelizi, D., Trursz,T., Bertoglio, J. 1987.Epstein-Barrviruscontaining B-cell line produces an interleukin 1 that it uses as a growth factor. Proc. Natl. Acad.Sci. USA84: 804 89. Durum, S. K., Schmidt, J. A., Oppenheim,J. J. 1985. Intedeukin 1: Animmunologicalperspective. Ann. Rev. Immunol.3:263 90. Oppenheim, J. J., Kovacs,E. J., Matsushima,K., Durum,S. K. 1986. There is more than one interleukin 1. Immunol.Today7(2): 91. Le, J., Vilcek, J. 1987.Tumornecrosis factor andinterleukin 1: cytokineswith multipleoverlappingbiologicalactivities. Lab.Invest. 56(3):234 92. Lomedieo, P. T., Gubler, U., Hellmann,C. P., Dukovich,M., Giri, J. G., Pan, Y. E., Collier, K., Semionow,R., Chua, A. O., Mizel, S. B. 1984. Cloning and expression of routine interleukin-1 eDNA in Escherichia coli. Nature312:458 93. March,C. J., Mosley,B., Larsen, A., Cerretti, D. P., Bradet, G., Price, V., Gillis, S., Henney,C. S., Kronheim, S. R., Grabstein,K., Conlon,P. J., Hopp, T. P., Cosman, D. 1985. Cloning, sequenceandexpressionof two distinct humaninterleukin-1 complementary DNAs.Nature 315:641 94. Auron, P. E., Webb,A. C., Rosenwasser, L. J., Mucci,S. F., Rich, A., Wolff, S. M., Dinarello, C. 1984. Nucleotide sequence of humanmonocyte interleukin 1 precursor eDNA. Proc. Natl. Acad.Sci. USA81:7907 95. Acres,R. B., Larsen,A., Conlon,P. J. 1987. IL 1 expression in a clone of humanT cells. J. Immunol.138:2132

Annual Reviews

Annu. Rev. Immunol. 1988.6:439-464. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

462 TANIGUCHI 96. Nedwin,G. E., Naylor, S. L., Sakaguchi, A. Y., Smith,D., Jarrett-Nedwin, J., Pennica,D., Goeddel,D. V., Gray, P. W. 1985. Human lymphotoxin andtumornecrosis factor genes:structure, homology and chromosomal localization. Nucl. Acids Res. 13(17): 6361 97. Spies, T., Morton,C. C., Nedospasov, S. A., Fiers, W.,Pious, D., Strominger, J. L. 1986.Genesfor the tumornecrosis factors alpha andbeta are linked to the humanmajor histocompatibility complex. Proc. Natl. Acad. Sci. USA83: 8699 98. Pennica, D., Goeddel, D. V. 1987. Cloning and characterization of the genes for human and murine tumor necrosis factor. SeeRef. 49, 13:163 99. Efrat, S., Kaempfer,R. 1984. Control of biologically active interleukin 2 messengerRNAformation in induced humanlymphocytes. Proc. NatL Acad. Sci. USA81:2601 100. Mitchell, R. L., Zokas,L., Schreiber, R. D., Verma,I. M.1985. Rapidinduction of the expression of proto-oncogene fos during human monocytic differentiation. Cell 40:209 101. Shaw, G., Kamen, R. 1986. A conserved AU sequence from the 3’ untranslated region of GM-CSF mRNAmediates selective mRNA degradation. Cell 46:659 102. Caput, D., Beutler, B., Hartog, K., Thayer,R., Brown-Shimer, S., Cerami, A. 1986. Identification of a common nucleotide sequencein the 3’ untranslated region ofmRNA moleculesspecifying inflammatorymediators. Proc. Natl. Acad. Sci. USA83:1670 103. Nagata, S., Tsuchiya, M., Asano,S., Yamamoto,O., Hirata, Y., Kubota, N., Oheda, M., Monura, H., Yamazaki,T. 1986. The chromosomal gene structure and two mRNAs for humangranulocyte colony-stimulating factor. EMBO J. 5(3): 575 104. Wong,G., Temple,P. A., Leary, A. C., Witek-Giannotti, J. S., Yang,Y.-C., Ciarletta, A. B., Chung,M., Murtha, P., Kriz, R., Kaufman, P. J., Ferenz,C. R., Sibley, B. S., Turner,K.J., Hewick, R. M., Clark, S. C., Yanai,N., Yokota, H., Yamada, M., Saito, M., Motoyoshi, K., Takaku, F. 1987. HumanCSF-1: Molecular cloning and expression of 4-KbcDNAencodingthe humanurinary protein. Science 235: 1504 105. Rajavashisth, T. B., Eng, R., Shadduck, R. K., Waheed,A., BenAvram,C. M., Shively, J. E., Lusis,

A. J. 1987. Cloningandtissue-specific expression of mousemacrophagecolony-stimulating factor mRNA. Proc. Natl. Acad. Sci. USA84:1157 106. Stasny, P., Rosenthal,M., Andreis,M., Ziff, M. 1975. Lymphokinesin the rheumatoidjoint. Arthritis Rheum.18: 237 107. Egeland, T., Lund, H. 1987. Immunoregulatory lymphokinesin rheumatoid joint. Scand.J. Immunol.25:101 108. Buchan,G., Barrett, K., Taniguchi,T., Maini, R., Feldmann,M. 1987. Detection of activated T cell productsin the rheumatoid joint using cDNAprobes to IL-2, IL-2 receptor and ~-IFN. Lancet.In press 109. Zoumbos,N. C., Gascon,P., Djeu, J. Y., Young,N. S. 1984. Interferon is a mediatorof hematopoieticsuppression in aplastic anemiain vitro andpossibly in vivo. Proc.Natl. Acad.Sci. USA82: 188 110. Leung, D. Y. M., Geha, R. S., Newburger, J. W.,Burns,J. C., Fiers, W., Lapierre,L. A., Pober, S. A. 1986.Two monokines,interleukin 1 and tumor necrosis factor, render cultured vascular endothelial cells susceptible to lysis by antibodies circulating during Kawasakisyndrome.J. Exp. Med.164: 1958 111. Sporn, M.B., Roberts, A. 1985. Autocrine growthfactors and cancer. Nature 313:745 112. Klein, G., Klein, E. 1986. Conditioned tumorigenicityof activated oncogenes. CancerRes. 46:3211 113. Uchiyama,T., Yodoi,J., Sawada,K., Takatsuki, K., Uchino,H. 1977. Adult T-cell leukemia: clinical and hematologicfeatures of 16cases. Blood50: 481 114. Broder, S., Gallo, R. C. 1985. Human T-cell leukemiavirus (HTLV):a unique familyof pathogenicretroviruses. Ann. Rev. Immunol.3:321 115. Wong-Staal, F., Gallo, R. C. 1985. HumanT-lymphotropic retroviruses. Nature 317:395 116. Yoshida, M., Seiki, M. 1987. Recent advancesin the molecularbiology of HTLV-I:Trans-activation of viral and cellular genes. Ann.Rev. Immunol.5: 541 117. Gottenberg, J. E., Ruscetti, F. W., Mier, J. M., Gazdar,A., Gallo, R. C. 1981. Humancutaneous T cell lymphomaand leukemiacell lines produce andrespondto T cell growthfactor. J. Exp. Med. 154:1403 118. Hattori, T., Uchiyama,T., Toibana, T., Takatsuki, K., Uchino, H. 1981.

Annual Reviews

Annu. Rev. Immunol. 1988.6:439-464. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

CYTOKINEGENE REGULATION Surface phenotypeof Japaneseadult Tcell leukemia cells characterized by monoclonalantibodies. Blood 58: 645 119. Depper,J. M., Leonard,W.J., Kronke, M., Waldmann,T. A., Greene, W. C. 1984. Augmented T cell .growthfactor receptor expression ~n HTLV-1infected humanleukemic T cells. J. Immunol. 133:1691 120. Sodroski, J. G., Rosen,C. A., Haseltine, W.A. 1984. Trans-actingtranscriptional activation of the long terminal repeat of humanT lymphotropic viruses in infected cells. Science225: 381 121. Felber, B. K., Paskalis, H., KleinmanEwing,C., Wong-Staal,F., Pavlakis, G. 1985. The pXprotein of HTLV-1 is a transcriptional activator of its long terminal repeats. Science229:675 122. Fujisawa,J., Seiki, M., Kiyokawa, T., Yoshida, M. 1985. Functional activation of the long terminal repeat of humanT-cell leukemiavirus type 1 by a trans-actingfactor. Proc.Natl. dcad. Sci. USA82:2277 123. Maruyama,M., Shibuya, H., Harada, H., Hatakeyama, M., Seiki, M., Fujita, T., Inoue, J., Yoshida,M., Taniguchi, T. 1987. Evidencefor aberrant activation of the interleukin-2 autocrine loop by HTLV-l-encoded p40x and T3/Ti complextriggering. Cell 48: 343 124. Shibuya, H., Harada, H., Maruyama, M., Fujita, T., Seiki, M., Inoue, J., Yoshida, M., Hatakeyama,M., Taniguchi, T. 1987. Twostep activation of the interleukin-2 autocrine loop may be involved in ATLdevelopment. In MolecularBasis of Lymphokine Action, ed. S. Cohen,C. Pierce, D. Webb.New York: Humana.In press 125. Cross,S. L., Feinberg,M.B., Wolf,J. B., Holbrook,N. J., Wong-Staal,F., Leonard, W. J. 1987. Regulation of the humaninterleukin-2 receptor ~ chain promoter: Activation of a nonfunctional promoter by the transactivator gene of HTLV-1.Cell 49:47 126. Inoue, J., Seiki, M., Taniguchi,T., Tsuru, S., Yoshida,M.1986. Induction of interleukin 2 receptor gene expression by p40X encoded by human T-cell leukemiavirus type I. EMBO J. 5:2883 127. Uchiyama,T., Hori, T., Tsudo, M., Wano,Y., Umadome, H., Tamori, S., Yodoi, J., Maeda, M., Sawami, H., Uctaino,H. 1985. Interleukin-2 receptor (Tac antigen) expressedon adult

463

cell leukemiacells. J. Clin. Invest. 76: 446 128. Maeda,M., Shimizu, A., Ikura, K., Okamoto,H., Kashihara, M., Uchiyama,T., Honjo, T., Yodoi, J. 1985. Origin of human T-lymphotrophic virus I-positive T cell lines in AdultT Cell Leukemia:AnalysisofTcell receptor gene arrangement. J. Exp. Med. 162:2169 129. Arima, N., Daitoku, Y., Ohgaki, S., Fukumori,J., Tanaka, H., Yamamoto, Y., Fujimoto, K., Onoue, K. 1986. Autocrinegrowthof interleukin-2-producingleukemiccells in a patient with Adult T Cell Leukemia.Blood 68(3): 779 130. Yamada,G., Kitamura, Y., Sonoda, H., Harada,H., Taki, S., Mulligan,R. C., Osawa, H., Diamantstein, T., Yokoyama,S., Taniguchi, T. 1987. Retroviral expressionof the humanIL2 genein a murineT cell line results in cell growth autonomy and tumorigenicity. EMBO J. 6(9): 2705 131. Duprez, V., Lonoir, G., DautryVarsat, D. 1985. Autocrine growth stimulation of a humanT-cell lymphoma line by interleukin2. Proc.Natl. Acad. Sci. USA82:6932 132. Blazar, B. A., Sutton, L. M., Strome, M. 1983. Self-stimulating growthfactor productionby B-cell lines derived from Burkitt’s lymphomasand other lines transformedin vitro by EpsteinBarr Virus. CancerRes. 43:4562 133. Gordon,J., Ley, S. C., Melamed,M. D., Aman,P., Hughes-Jones, N. C. 1984. Soluble factor requirementsfor the autostimulatory growth of B lymphoplasts immortalized by EpsteinBarr Virus. J. Exp. Med.159:1554 134. Pettenati, M. J., LeBeau, M. M., Lemons, R. S., Shima, E. A., Kawasaki,E. S., Larson, R. A., Sherr, C. J., Diaz, M.O., Rowley,J. D. 1987. Assignment of CSF-1 to 5q33.1: Evidencefor clustering of genesregulating hematopoiesis and for their involvement in the deletion of the long arm of chromosome 5 in myeloid disorders. Proc. Natl. Acad.Sci. USA 84:2970 135. Heard,J. M., Fichelson, S., Sola, B., Martial, M.A., Varet, B., Levy,J. P. 1984. Multistep virus-induced leukemogenesis in vitro: description of a modelspecifyingthree steps within the myeloblastic malignantprocess. Mol. Cell. Biol. 4:216 136. Ymer,S., Tucker,Q. J., Sanderson,C. J., Hapel, A. J., Campbell, H. D., Young, I. G. 1985. Constitutive

Annual Reviews 464

TANIGUCHI

Annu. Rev. Immunol. 1988.6:439-464. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

synthesis of interleukin-3 by leukemia Autocrine growth induced by srccell line WEHI-3B is due to retroviral related oncogenes in transformed insertion near the gene. Nature 317: chickenmyeloidcells. Cell 39:439 255 139. Graf, T., Weizsaecker,V. F., Grieser, 137. Lang, R. A., Matcalf, D., Gough,N. S., Coil, J., Stehelin,D., Patschinsky, M., Dunn, A. R., Gonda,T. J. 1985. T., Bister, K., Bechade,C., Calothy, Expressionof a hematopoieticgrowth G., Leutz, A. 1986.v-mil inducesautofactor cDNA in a factor-dependentcell crine growth and enhanced tumoline results in autonomous growthand rigenicity in v-myc-transformedavian tumorigenicity.Cell 43:531 macrophages.Cell 45:357 138. Adkins,B., Leutz, A., Graf, T. 1984.

Annu. Rev. Immunol. 1988.6:439-464. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

Annu. Rev. Immunol. 1988.6:439-464. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

Annual Reviews www.annualreviews.org/aronline Ann. Rev. Immunol. 1988. 6 : 46543 Copyright © 1988 by Annual Reviews Inc. All rights reserved

Annu. Rev. Immunol. 1988.6:465-483. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

UNIQUE TUMOR-SPECIFIC ANTIGENS Hans Schreiber, Patricia and Hans J. Stauss

L. Ward, Donald A. Rowley

Division of Biological Sciences, TheUniversityof Chicago, Chicago,Illinois 60637 INTRODUCTION Oneof the oldest and most important questions in cancer immunology concernsthe existenceof tumor-specificantigens (1). Theexistenceof such antigens wouldrepresent a qualitative difference betweena malignantand a normalcell, and the antigenwouldbe clinically useful in tumordiagnosis, prophylaxis, and therapy. The nature of tumor antigens mightalso give us a clue to the causes of the malignantprocess itself. However,despite vast efforts by immunologistsduring the last century, the existence of tumor-specificantigens in the majority of cancers remainsunproven.This absence of proof despite extensive effort has commonly led to the view that tumor-specificantigens simplydo not exist. However, critics usually underestimate the considerabletechnical difficulties andpitfalls of experimentsdesignedto provethe existenceof tumor-specificantigens, especially onhuman cancercells. In this review,therefore, wetry to evaluatecritically the available evidencefor the existenceof tumor-specificantigens, andwe also discuss the majorproblemsthat havehamperedrapid progress in this important field of tumor immunology. EVIDENCE SPECIFIC

FOR THE EXISTENCE OF TUMORANTIGENS ON MURINE TUMORS

Since the turn of the century (1), transplantation experimentshavebeen performed to determine whether it was possible to immunizeagainst cancer. Thoughthese early experimentsseemedto suggest that immunization against cancer waspossible (2-4) the results weredubious, since 465 0732-0582/88/0410-0465502.00

Annual Reviews

Annu. Rev. Immunol. 1988.6:465-483. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

466

SCHREIBER ET AL

outbred mice or rats were used. Later it was realized that immunization with tumorsalso immunizedagainst normaltissues of the donor and that normal tissues of the donors also immunizedagainst the tumors. While these transplantation studies eventuallyled to the discoveryof the major histocompatibility complex(5, 6) and other importantantigens, the idea of tumor-specificantigens cameinto disrepute. Resistanceto tumortransplants of a chemicallyinducedsarcomain syngeneicmicewasfirst demonstrated in 1943(7). But it was not until the 1950s that morecomplete experimentsusing inbred mouseand rat strains (8-13) providedunequivocal evidencefor the existenceof tumor-specificantigens, i.e. miceor rats first immunizedwith a particular tumor and then challenged with the sametumorshowedprotective immunity.In particular, the experimentsof Prehn & Main(10) in 1957madeit highly likely that the antigens were tumor-specificand werenot due to residual heter0zygosityin the inbred mousestrain used (14). Theyshowedthat normaltissues of the mouse tumororigin did not immunizeother mice against the tumor nor did the tumorimmunizeagainst skin grafts from the mouseof tumororigin. While immunization with tumortissue did cause protective immunity,final proof camefrom experimentsof Klein et al (11) in 1960 showingthat tumorspecific resistance against methylcholanthrene-induced sarcomascan be induced in the primary autochthonoushost. The immunologicresistance that can be induced against chemically induced sarcomas was usually relative rather than absolute and broke downwhenthe numberof tumor cells used wasincreased. In contrast, murinetumorsinducedby ultraviolet light maybe muchmoreantigenic (15) so that very large transplants tumors into normalsyngeneicmice do not growprogressively (e.g. > 20 3 tumorfragments). Whilesuch large tumorloads are rejected by the 1 mm normalmice, small transplants will growprogressively in nudemiceJ

~ Althoughtumor-specific transplantation antigens are most clearly associated with chemically and physically induced cancers, such antigens are also knownto exist on some other "spontaneous" cancers (16-18). Nevertheless, "spontaneous" cancers appear in general be muchless immunogenicin transplantation experiments than are experimentally induced cancers, and this has been knownfor decades (8, I0). This lower immunogenicity may due in part to the longer latency period of these tumors which leaves more time for extensive immunoselection. However, even though spontaneous tumors frequently fail to induce an active protective immunity,theY maystill possess tumor-specific surface antigens (19) that can serve as targets for diagnosis and treatment. Unfortunately, the interpretation of several studies analyzing the antigenicity of spontaneous tumors is complicated by the fact that the tumors used had been serially transplanted and mayhave, therefore, been immunoselected for antigen loss variants (20, 21), since this mayoccur during a single passage (22, 23). Because of space limitation, the various factors controlling the immunogenicity of tumors (13, 24, 25) cannot be discussed here in any detail.

Annual Reviews UNIQUE TUMOR-SPECIFIC ANTIGENS

Annu. Rev. Immunol. 1988.6:465-483. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

ANTIGENIC DIVERSITY: SHARED ANTIGENS

467

UNIQUE VERSUS

Oneof the objectives of experiments using chemically induced murine tumors was to search for antigens on tumors that were not only tumorspecific but also were shared amongindependently induced tumors, so that such antigens could be used for diagnostic or therapeutic purposes. Instead, transplantationexperimentsrevealedthat the tumor-specificrejection antigens wereusually individually tumor-specific, i.e. uniquefor a particular tumor even when compared to other tumors of the same histologic type inducedin the sameorgan system by the samecarcinogen (Table1). Whilecross-reactivity betweenindependently induced tumors has occasionally beenreported (for reviewsee 26), Basombrio (27, 28) suggested that the diversity of tumor-specifictransplantation antigens maybe very large. In fact, Basombrio’ssearch for common antigens among10 methylcholanthrene-inducedsarcomasrevealed no reproduciblecross-protection, in 90 tests for cross-protection using micefirst challengedwith one and then with another syngeneicsarcoma(Figure 1); protection wasuniquely specific for the particular tumor used for immunization.Evenmultiple immunizationsusing a mixture of four tumors and challenging with a single different tumorrevealedno cross-protectionin 13 out of 14 experiments. Onemixture alone gave reproducible cross-protection against one other tumor but even then, not against four other tumors tested. The sporadic findings of cross-reactivity by different authors suggestthat the size of the antigenic repertoire of uniquetumor-specifictransplantation

Table 1 Characteristics

of unique tumor-specific antigens

1. Specific for an individual tumor whencompared to other tumors --of the same histologic type --induced in the same organ system --by the same carcinogen --in the same mousestrain 2. Defined by tumor transplantation experiments using syngeneic hosts (not possible in humans) 3. Found in tumors induced by chemical and physical carcinogens 4. Verylarge antigenic diversity 5. Presumablynot caused by clonal amplification of preexistent antigens 6. Presumably not present on normal cells (but no final proof using in vitro probes and autologous controls)

Annual Reviews

468

SCHREIBER ET AL

Annu. Rev. Immunol. 1988.6:465-483. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Challenged with Tumor

E Figure 1 Demonstration of uniquely specific transplantation antigens on independently derived methylcholanthrene-induced sarcomas in mice of the same inbred strain. No reproducible cross-reactions were revealed upon screening for shared antigenicity. Solid squares: rejection of tumor inoculum. Hatched squares: unrepeatable rejections. Empty squares: combinations that showed no rejection. Modified from Basombrio & Prehn (28).

antigens maybe limited. However,it must be realized that weakresistance against challenge with-living tumor cells can sometimesbe induced with unrelated tumors or even normal tissues (11). This may be caused nonspecific bolstering of immuneresponses to specific antigens (11). Cross-protection is also observed in the model of UV-inducedmurine tumors; for example, immunization with highly immunogenic regressor tumors can lead to protective immunity against challenge with an independently induced, less immunogenicprogressor tumor (29). Again, it possible that this cross-protective effect is caused by nonspecific bolstering of immuneresponses to specific antigens, since unlike immunity that is specific for individual tumors, this cross-protection is usually sensitive to low doses of gammaradiation and fades with time (30). As noted Brent & Medawar(31), immune responses become radioresistant once immunization has occurred. Although "cross-reactivity" among independent UV-inducedtumors has also been found to exist in CTLgenerated in bulk cultures under certain experimental conditions (22, 29), analysis

Annual Reviews

Annu. Rev. Immunol. 1988.6:465-483. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

UNIQUE TUMOR-SPECIFIC

ANTIGENS

469

of cross-reactivityat the clonal level usinglimitingdilution (22) indicated clearly that this reactivity wasnot the result of activationof clonesdirected against a putative common UVantigen but was due rather to polyclonal activation of CTLthat can occur in culture. Thus at present, transplantation experiments with chemically or physically induced murine tumorsprovide clear evidence for the existence of strong unique tumorspecific transplantationantigens. Thereis, however,little if any evidence for the existenceof strong cross-protectivebut tumor-specificantigensthat would be commonlyexpressed by independently induced tumors. [The weakcross-protective immunityoccasionally observedmayalso be due to the expressionof sharedfetal antigens. Thepresenceof these antigens that are not tumor-specific on humanand animal tumors has been reviewed extensively(32).] Whiletransplantation assays have failed to bring clear evidence for antigens that are tumor-specific yet shared amongtumors, serological studies on murineleukemiashave led to the discovery of two classes of shared antigens, the TLantigen (33) and the viral surface glycoprotein antigens. Apparently, all mousestrains have the genes encodingthese antigens, but the regulation of their expression is strain specific. For example, somemousestrains express TL antigens during normalthymic developmentwhile these antigens are never expressed in normal thymocytes of other mousestrains. However,leukemiccells frequently express TLantigens independentof whetherthe leukemiaarose in a TL-positive or in a TL-negativemousestrain (for review see 34). Althoughthese antigensare not tumor-specificin the strictest sense, they are tumor-specific in those mousestrains that fail to derepress TLgenes in normalcells duringfetal andadult life. Therefore,one mightexpectthat these antigens still serve as tumor-specificrejection antigens or could be targets for immunotherapy. However, immunization with the TL antigen on the leukemiccells does not generate transplantation resistance. Theabsence of protection is due to a rapid reversible loss of TLantigen from the leukemiccells, a phenomenon that led to the discoveryof antigenic modulation (35). Similar to the TLantigen, viral surface glycoproteinsare also not the targets for protective tumor immunityon chemically induced sarcomaseven though high titers of antibody against such antigens are produced(36). A third class of shared antigens maybe products of activated cellular oncogenes,e.g. the neu-1antigen(37-39). Productsof cellular oncogenesmaybe present on fetal cells, activated adult cells, and sometimesin lesser amountson resting adult cells. Althoughantibodies directed against such oncogene-encoded antigens mightlead to sometemporary inhibition of tumor growth, these antigens again are apparently not tumor-specificantigensin the strictest sense.

Annual Reviews 470

SCHREIBER ET AL

Annu. Rev. Immunol. 1988.6:465-483. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

ORIGIN OF DIVERSITY: MUTATION, OR CLONAL AMPLIFICATION

ACTIVATION,

Althoughuniquetumor-specific transplantation antigens were discovered three decadesago, the molecularnature and genetic origins of these antigenshaveremainedobscure,andthe basis for their large antigenicdiversity has remainedan enigma.Possibilities are (i) preexistingantigenicdiversity in normalsomaticcells giving rise to the cancer(40), (ii) derepression activation of fetal antigens (41, 42), (iii) derepressionor mutationof class I genes(43, 44), (iv) antigens linked to the immunoglobulin heavy chain (Igh) locus (45), (v) antigens related to recombinantmurine viruses (46, 47), (vi) antigensrelated to heat shockproteins (48), and epigeneticerrors in the assemblyof cell surface recognitionsites (49). Since most (and possibly all) chemicaland physical carcinogens are mutagens,it is quite conceivablethat individually distinct transplantation antigens are caused by carcinogen-inducedmutations in cellular genes. Alternatively,the diversity of tumor-specificantigens mayalready preexist in the normalcells, andthe carcinogenmaylead to the clonal amplification of single cells expressinga particular antigen. Accordingto the clonal amplificationhypothesis(40), eachnormalprecursorcell contains different antigens, these are, however,not in sufficient quantity to be recognizedby the immune systemuntil clonal amplification leads to a tumor.Anexample of an analogoussituation are the idiotypes on B or T cell malignancies that are present on a normallymphocyte clone but only on such a restricted numberof normal lymphocytesthat the immunesystem does not develop toleranceto suchidiotypes(50). In this situation the idiotypecanstill serve as target antigens for diagnosis andtherapy (50-52) or play a role in the proposedreceptor-mediatedleukemogenesis(53, 54). Twoindependentstudies have approachedthe question of clonal amplification experimentally(55, 56). In one study(55), Balb/c3T3cells cloned in vitro weremalignantlytransformedby methylcholanthrenein diffusion chambersin the abdomenof mice. Transplantation experiments showed that tumors developingfrom the malignanttransformants had individually distinct antigens eventhoughthey all had beenderivedfromthe samecell. The second study (56) reported that tumors induced by methylcholanthrenein vitro in progenyof cells froma single fibroblast clone also had individually distinct antigens. It remainedunclear, however,whether these cell lines also had uniquetumor-specifictransplantation antigens, since they were not tested in vivo. Eventhoughthese data suggest that carcinogensmaycause the appearanceof a newantigen ("neoantigen") the tumorcell, the mechanism maystill be carcinogen-inducedactivation of a preexistent silent gene. For example, somegene families (57-59)

Annual Reviews

Annu. Rev. Immunol. 1988.6:465-483. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

UNIQUETUMOR-SPECIFIC ANTIGENS 471 harbor a large number of silent distinct genes that could be randomly activated by the carcinogen, and this may cause considerable antigenic diversity. An example of a nonrandomactivation of a preexistent gene is the appearance of the TLantigen in leukemic cells of TL negative strains (for review see 60). Whyin this case only the TLgene is selectively and consistently activated (and apparently no other silent class I gene of the MHC complex) is unclear. Certainly, a random activation of other preexistent and silent class I genes could lead to considerable antigenic diversity even without recombination. Based on serological (47) and biochemical (46) studies, activation recombination events in the family of gp70 genes have been proposed as another mechanismleading to unique tumor-specific antigens. However, no evidence suggests that any of these gp70 molecules actually serve as unique tumor-specific rejection antigens. Thus, at present there is no conclusive evidence that tumor-specific transplantation antigens are caused by somatic mutations of structural genes, despite the well-accepted fact that most carcinogens are mutagens. Only for one tumor (44) has been possible to document that changes in the primary sequence of DNA occurred in the tumor and were not present in the mousestrain of tumor origin. However, because of the lack of normal control cells from the original host that gave rise to this tumor, it cannot be concluded with absolute certainty that the antigen did not preexist on normal cells of the original host, nor that it was due to a germline mutation rather than to a carcinogen-induced somatic mutation. Further studies using tumors for which autochthonous normal control cells are available will be needed to answer this question conclusively.

RELATIONSHIP

TO MALIGNANT BEHAVIOR

That the unique tumor-specific transplantation antigens appear to be the only changes presently knownto be tumor-specific led to muchspeculation as to whether a causal relationship might exist between tumor-spec.ific antigens and the expression of malignant behavior. Boyse (49) suggested that tumor antigens mayrepresent heritable alterations in cell-cell recognition sites, the dysfunction of whichmayresult in abnormalor invasive growth. Similarly, a completeloss of such cell-cell recognition sites could also cause the malignant phenotype, with the added consequence that the cancer may appear "nonantigenic." Considering the lack of selectivity of the mutational effects of chemical or physical carcinogens, a causal relationship of tumor antigens to malignant behavior might appear Unlikely since randomchanges would only very rarely hit a gene important for the establishment of malignant behavior. However, we must also con-

Annual Reviews

Annu. Rev. Immunol. 1988.6:465-483. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

472

SCHREIBER ET AL

sider that the mutationalevents causedby chemicalor physical carcinogens maygive the target cell a combinationof advantagesand disadvantages. Manyof the mutational changes randomlyinduced by carcinogens are probablya disadvantagefor the cell and are, therefore, selected against during the clonal evolution leading to cancer (61-65). Simultaneously, there maybe selective retention of specific mutationalchangesthat favor the malignant process. Onesuch examplemight be the apparently favorable mutationin a cellular protooncogenein carcinogen-inducedtumors (66). Another examplemight be the proposed receptor-mediated leukemogenesis(54). It has beenarguedthat the antigenicdiversity of tumor-specificantigens evenin closely related tumorsmakesit unlikely that these antigens represent changes of the same receptors. However,weknowthat enormous antigenic diversity can be generatedby genetic alterations of evena very smallregionof a single molecule(46, 58, 59). Finally, experimentalstudies (44, 45) haveimplicatedtwomajortypes of cell-cell interaction molecules, namelythose molecules encoded by the immunoglobulin(45) and major histocompatibility gene clusters (44), both of whichbelong in the same supergenefamily encodingcell surface recognition molecules(57-59). addition, there is another group of normal genes, the so-called protooncogenes,that has been proposed to play a critical role in normal growthand developmentand in cancer (67, 68). Althoughthe products protooncogenes often are intracellular molecules,these productsmaystill appearas peptides on the outside of the cell after intracellular enzymatic cleavage (69, 70) and be recognized as tumor-specific antigens. Such possibility wassuggestedby the finding (71, 72) that the SV40geneencoding the T antigen (a nuclear protein knownto be critical for establishing and maintaining the malignant phenotype)also causes the appearanceof a cell surface antigen on SV40-transformed cells for MHC class 1-restricted CTLrecognition. Thus, the possibility exists that these protooncogenes whenmutatedby the carcinogenmightlead not only to the expression of malignancybut also to the expression of tumor-specific antigens. At present, however,wedo not knowwhether any or howmanyof the tumorspecific surface alterations described by immunologistsas uniquetumorspecific antigens are causally related to the malignantphenotype. IN VITRO PROBES Oneof the major stumbling blocks of tumor immunology is the virtual absenceof reliable probes that are tumor-specific, permanent,and transferable betweenlaboratories. Nodoubt, the availability of such in vitro probesis almosta prerequisitefor studies trying to determinethe molecular

Annual Reviews

Annu. Rev. Immunol. 1988.6:465-483. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

UNIQUE

TUMOR-SPECIFIC

ANTIGENS

473

nature and genetic origins of tumor-specific antigens. However,despite serious efforts by numerouslaboratories including ours, there has been almost no success in generating monoclonalantibodies specific for any of the tumor-specificantigens defined by transplantation assays. Thedifficulty is strongly reminiscent of that in generating monoclonalantibody probes against minor histocompatibility antigens that are also solely defined by transplantation assays (73). In the one tumor whereit has beenpossible to generate tumor-specificmonoclonalantibodies, the tumor antigen was found to be a novel (mutated)major histocompatibility class I antigen. Since it has been shownthat monoclonalantibodies can be raised to normalMHC class I antigens, this mayexplain the success in this one instance, and it also maysuggest that tumor-specificantigens on other tumorsmayoften not belongin the family of MHC class I molecules. However,this deductionmaybe incorrect, since other mutantH2products are knownto give rise to strong transplantation immunitywhile not stimulating an antibodyresponse(74). Cytolytic T cell clones mayalso serve as specific and permanentprobes for tumor-specific antigens. AlthoughCTLcannot be used for direct isolation of the tumor.antigen, they can still be very usefulfor definingthe genetic origin of tumorantigens. For example,tumor-specific CTLclones could be used to select for antigen loss variants caused by insertional mutagenesis.As suggestedby analogousexperiments(75), the isolation such variants could lead to the rapid isolation of the genes encoding for tumor-specific antigens. Tumor-specific CTLcan be cloned and so represent permanent,transferable, highly specific probesfor uniquetumorspecific antigens (Figure 2). In addition, CTLprobescan be used to define a uniquetumor-specificantigen as a rejection antigen. This can be done by selecting for tumorantigen loss variants with the CTLin vitro and by testing the variants subsequentlyin vivo for growth(Figure 3). CTLclones appear to be mucheasier to generate than tumor-specific monoclonalantibodies. For example,wehave been able to generate CTL against several tumor-specific antigens on UV-induced tumors, while we were unable to generate monoclonalantibodies with the samereactivity. Possibly tumor-specificantigens are located primarily intracellularly, so that antigenic peptides generatedby intracellular enzymaticcleavage and movedto the cell surface in association with MHC are recognizedby CTL clones but not by the immunoglobulin receptor of B cells. Examplesof such a mechanismhave been mentionedin the previous section. If this mechanismis relevant and if unique tumor-specific antigens are indeed primarily intracellular proteins, then twofurther deductionscan be made. First, it maybe easier to generate tumor-specific monoclonalantibodies to intracellular than to cell surfaceantigens. Second(and this is the more

Annual Reviews 474

SCHREIBER

ET AL I

Annu. Rev. Immunol. 1988.6:465-483. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

8(]

~" 2240 ~ II 0462 v 918 o 1591 ¯ 1465 ¯ 3152 n 1316 ¯ 1130 ¯ HLF o MSFI9

0.3:1 0.6:1 0.2:1 2.5:1 Effector-to-Torgel Cell Rotio Fiyure2 Specificity of a 2240CTLline generatedby syngeneicimmunization withlive tumorcells.

fundamental aspect of such a mechanism), CTLmay be able to eliminate cells expressing mutated intracellular molecules following carcinogen exposure. It is important to note that CTLprobes for tumor-specific antigens have not been tested for specificity using autochthonousnormal cells as negative control targets, because nontumor control cells have not been isolated concurrently with the malignant cells from the same mousedeveloping the tumor. It is obvious that such controls are required for defining the genetic origin of tumor-specific antigens, just as the ultimate evidence for the existence of tumor-specific transplantation antigens depended upon the use of the autochthonous host. Without such autochthonous control cells, it will be virtually impossible to exclude the possibility that residual heterozygozity (14) or somatic mutation in the mouse of tumor origin was responsible for the apparent tumor-specificity.

Annual Reviews UNIQUE TUMOR-SPECIFIC ANTIGENS

475

I anti-A "~, , ce(~| c Yt°l~,’nCe A

B

Annu. Rev. Immunol. 1988.6:465-483. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Parent

Regressor

B

~/~"--"~.

Selection in vitro

Variant

Progressor

Figure 3 Schemeof experiment (80) to test the relevance of a CTL-defined unique tumorspecific antigen for tumorrejection in vivo. Loss of the Aantigen leads to variants showing progressive tumor growth in normal mice while the reciprocal variants selected for loss of the B antigen are still rejected by the normal host. Reprinted from Koeppenet al (25) with kind permission from the publishers.

THE METHYLCHOLANTHRENE-INDUCED TUMOR METH A The unique tumor antigens on the methylcholanthrene-induced sarcoma Meth A have been analyzed extensively (76). Furthermore, several quite interesting differences exist between the MethA tumor and the UV-induced tumor 1591. In contrast to the tumor 1591-RE, variants that have lost transplantation antigens have not been isolated from the Meth A tumor. This finding has been taken to suggest that the tumor-specific rejection antigens on the Meth A tumor may be required for the expression of malignant behavior. Also, in contrast to the tumor 1591-RE,it appears to be very difficult or impossible to generate tumor-specific CTLto Meth A, suggesting that rejection of the Meth A tumor by immunemice does not + helper T cells. Immunity involve Lyt2+ cytolytic T cells but rather L3T4 mediated by helper T cells maynot be effective in selecting antigen loss variants, a conclusion suggested by results in the 1591 tumor model where only CTLbut not T helper cell-recognized antigens are lost (30). In the Meth A tumor model, it has been possible to generate tumor-specific antisera following extensive absorption. With the use of these antisera and microcell chromosometransfer, it has been possible to map this serologically defined antigen to genes linked to the immunoglobulinheavy chain (Igh) locus (45). It is not clear howthis MethA-specific antigen relates to the MethA tumor-specific rejection antigen. Later studies, therefore, used transplantation tests for analyzing fractions of MethA cell lysates to purify and identify the tumor-specific rejection (transplantation)

Annual Reviews

Annu. Rev. Immunol. 1988.6:465-483. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

476

SCHREIBER ET AL

antigen. This approachled to the isolation of a protein fromthe methylcholanthrene-inducedtumorthat could be used to immunizemice, specifically inhibiting the growth of the MethA tumor but not the growthof other methylcholanthrene-inducedtumors (48). The proteins that conferred transplantation immunityconsisted of two polypeptideisoforms of 84 and 86 kd, each of whichhad an identical NH2-terminalaminoacid sequence. Both polypeptides appeared homologousto the Drosophila83kd heat shock protein (48), suggesting that the purified MethA tumor antigen mayrepresent the 85-kd murineheat shock protein. This protein has also been found in other tumors and normal cells, and so far no alteration has beendetectedin the protein that accountsfor the specificity of protection caused by immunizingwith the Meth A protein. Using different techniques but the same MethA tumor, Srivastava et al (98) found a 96-kd glycoprotein that also acted as a MethA tumor-specific rejection antigen. Anothermethylcholanthrene-inducedtumor that was antigenically distinct fromMethA also had a 96-kdglycoproteinas unique tumor-specificrejection antigen, but again no differences haveyet been found betweenthe two 96-kd proteins to account for unique tumorspecificity. Furthermore,it is not clear whetherthe three different MethA antigens describedaboveare expressedindependentlyof each other since, unlike the 1591-REtumorsystem, variants selectively expressing one or the other antigen are not available. Nevertheless,it is quite conceivable that Meth A tumors also express multiple independent unique tumorspecific transplantation antigens, as wehaveshownpreviously(77) for the 1591-REtumor (see below). THE ULTRAVIOLET SKIN TUMOR 1591

LIGHT-INDUCED

Several aspects of the antigens on the UV-inducedtumor 1591have been reviewedbefore(25, 78, 79) andare not repeatedhere in detail. In contrast to the tumor MethA whichonly induces a relative (i.e. challenge dosedependent) protection by immunization, the UV-inducedtumor 1591 is rejected by normalyoungmice even whenmultiple large tumor fragments are transplanted. Highimmunogenicity is shared by several of the other UV-inducedtumors (15), including someof the tumors we have isolated recently (1984 to 1987, Patricia L. Ward,HansSchreiber, unpublished), whichsuggests that this is a common feature of UV-induced tumors. Tumor-specific CTLclones can be generated relatively easily and dependablyto at least someof the tumor-specificrejection antigens. Such CTLclones are exceedinglyspecific and reliable, and can be shared with other laboratories. Wehaveused such CTLclones (see Figure 4) to dissect

Annual Reviews

Annu. Rev. Immunol. 1988.6:465-483. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

UNIQUETUMOR-SPECIFICANTIGENS

477

Fi#ure4Schematicdiagramof the sequential selection wherebythe complexityof a unique tumor-specificantigencan be dissectedby generatingT cell lines. T cell lines are generated frommice immunizedwith in vitro selected tumorvariants. Reprintedfrom Koeppenet al (25) with kind permissionfromthe publishers.

the complexityof a uniquetumor-specific antigen. A syngeneicCTLline (called anti-A becauseit wasthe first isolated) waspreparedagainst 1591RE.The A- variants that were resistant to the anti-A CTLline after in vitro selection all grewout fromthe cultures used for in vitro selection. AnA- variant wasthen used to derive a secondCTLline ("anti-B") which to our surprise reacted with a different antigen present on the parental as well as on the variant tumorcell (80). Toour further surprise, wefound additional repeated cycles of selection that the tumorpossessedadditional specific C and Dantigens. All of these antigens wereuniquely1591tumor specific. It is importantto mentionthat all four tumor-specificantigens werealwayslost independentlyof each other (77). Expressionof multiple antigens as demonstratedfor 1591mayalso occur on the tumorP815(81) and in the waydiscussed aboveon MethA, suggesting that this maybe a common feature of tumors. Thoughit should be advantageousfor the host to respondto all unique antigens initially and simultaneously,unfortunately,this is not the case. For example,all variants expressingthe Aantigen elicit a specific CTL response limited or predominantlydirected to the A antigen even though other uniqueantigens are also present on these variants (82). Onlywhen the A antigen is lost is the next dominantantigen recognized(83). This peckingorder in the host response to multiple tumor-specific antigens causes sequential antigenic changesand loss, and it appears to permit selection of more malignant variants which then leads to tumor progression. For example, only 104 A-B-C-D-1591 variant tumor cells - cells kill micein 5 to 6 weeks; kill mice in about 3 weeks;107 AB÷C-D and A ÷ and or C ÷ parental tumorswill not growprogressively in normal mice at any testable dose. Someof the A-B-C-D-variants metastasize extensively to lymphnodes. Searchfor retained CTL-defined1591tumor-

Annual Reviews

Annu. Rev. Immunol. 1988.6:465-483. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

478

SCHREIBER ET AL

specific antigens on metastatic variants has been unsuccessful (30); however, this highly malignant tumor variant, as well as all other 1591 variants, expresses a unique 1591tumor-specific antigen that can be defined by helper T cells in vitro or by experiments measuring delayed type hypersensitivity in vivo. It is interesting that the highly metastatic variant which expresses only this antigen causes specific and protective immunityto any 1591variant that still expresses a CTL-definedantigen, but the metastatic variant cannot induce protective immunityto itself (30). To begin to determine the origins of the T cell-defined antigens on the 1591 tumor, we attempted to generate monoclonal antibodies that have the same reactivity pattern and also select for the same variants as with the specific CTLclones. Since the major immuneresponse to the tumorspecific transplantation andgensis T cell-mediated, difficulties in finding specific hybridomas must be expected. While hyperimmunization or isologous and heterologous immunization only yielded cross-reactive hybridomas, injection of syngeneic mice once with viable tumor cells, followed 4 weekslater by a single intravenous boost 3 days before fusion, elicited a successful response. The overall frequency of specific hybridomas was very low, i.e. about 1 in 2000 to 3000 hybridomacolonies. One hybridoma, CP28,produced antibody with reactivity to the 1591 tumor, which like the anti-A CTLprobe did not react with progressor variants selected by the host in vivo, nor did it react with normal embryonicor adult cells, or with 37 other syngeneic tumors (84, 85). The monoclonal antibody precipitated from the 1591 tumor a 45-kd molecule that was associated with a 12-kd molecule having the isoelectric point of f12 microglobulin (84). This and other evidence indicated that the 1591 tumor expressed a novel class I molecule, which might account for previously observed abnormal reac.tivity of alloantigen-specific antibodies with tumor cells. Althoughthese studies suggested the presence of altered MHC class I molecules on malignant cells (for review, see 86-90), the relationship of such antigens to the unique, i.e. individually specific antigens remainedinconclusive because of the absence of any in vitro probes. In contrast, there was a strong suggestion that the CP28antibody recognized the 1591 tumor-specific rejection antigen, a notion that was later proven correct by molecular cloning and transfection of the gene (44). Furthermore, only gene transfection experiments could show conclusively that the antibody and the CTLdefined epitopes were both present on the same molecule (44). Genecloning was used to determine whether the novel class I molecules on the 1591 tumor were due to mutational changes in MHCclass I genes or to activation of intact but previously silent genes of the MHC class I gene family (58). Three different MHC class I genes were isolated that could account for all the antibody defined novel MHCclass I determinants

Annual Reviews

Annu. Rev. Immunol. 1988.6:465-483. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

UNIQUE TUMOR-SPECIFIC

ANTIGENS

479

expressed by the 1591 tumor. Further analysis revealed that the three genes showedrestriction enzymefragment length polymorphismsthat were absent in the class I genes of normal C3Hmice (90). Thus the three novel class I genes probably did not preexist as intact genes in the genomeof the C3Hstrain from which the 1591 tumor originated. Since circumstantial evidence suggests that all three abnormal class I genes are present on a single chromosome,it is possible that residual heterozygosity accounts for the appearance of the antigens present on the 1591 tumor. However,this is unlikely because several equally immunogenic tumors isolated concurrently with the 1591 tumor exhibit normal isoenzyme patterns as compared with the present day C3Hmouse strain. These tumors do not share the novel class I antigens of 1591 and in fact all other UV-induced tumors tested so far do not show any detectable MHCclass I gene polymorphismby Southern blotting analysis. Further, in some very recent experiments, more than half of the isolated tumors were regressor tumors. Germline mutations are not thought to occur at this high frequency, nor should residual heterozygosity be so prevalent after several decades of inbreeding. While it appears that UVirradiation must be required for immunogenicity to occur, UVirradiation is immunosuppressive (88, 91-93) and possibly allows for the development spontaneous somatic MHCclass I mutants. The definitive answer to these important problems can obviously only be given once the genetic origin of other unique tumor-specific transplantation antigens is knownand tumor-specific in vitro probes are developed for tumors for which autochthonous control cells are available. HUMAN

TUMORS

As stated above, the only clear-cut evidence for the existence of truly tumor-specific antigens comes from transplantation studies using highly inbred mice. Since transplantation experiments cannot be done in humans, we do not knowwhether tumor-specific antigens exist in humantumors. It has been argued that "spontaneous" murine cancers represent model tumors that resemble more closely humanmalignancy (94). Since murine spontaneous tumors are usually much less immunogenic than murine chemically induced tumors, it would suggest that humancancers are not immunogenic. However, it is not clear at all how spontaneous murine tumors relate to humancancer (95). Certainly the evidence is overwhelming that most humancancer is induced by physical and chemical carcinogens (96). At present, it is clearly incorrect to assumethat tumor-specific antigens either exist or do not exist. Cancer immunologists appreciate very well the technical difficulties of generating in vitro probes to murinetumors

Annual Reviews 480

SCHREIBER ET AL

eventhoughtheymaybe exceedinglyantigenic,so it is reasonable to expect equivalentdifficulties in generatingprobesto antigenson human tumors. Oneapproachthat appearsto be mostpromisingis "autologoustyping" that uses reactionsbetweensera andtumorfromthe samepatient. Using this approach the existenceof individUallydistinct (unique)tumorantigens that are restrictedto autologous tumorcells hasbeensuggested,andstructural studies are underway to analyzethe origin(s) of suchantigens(97).

Annu. Rev. Immunol. 1988.6:465-483. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

ACKNOWLEDGMENTS

WethankCarolynHencefor excellent secretarial assistance. Theworkof H. Schreiber,P. L. Ward,D. A. Rowley,andH. J. Stausswassupportedby grants CA-19266,CA-22677,CA-37156,and AI-7090from the National CancerInstitute, NationalInstitutes of Health. Literature Cited 1. Ehrlich, P. 1909. Ober den jetzigen Stand der Karzinomforschung. Ned. Tijdschr. Geneeskd.5 (Pt. 1): 273-90 2. Dr. Uhlenhuth, Dr. Seiffert. 1925. Kritische ~bersicht fiber die Grundlagen der Immunit/it gegen transplantable Tumoren. Med. Klin. 21:57(~616 3. Woglom, W. H. 1929. Immunity to transplantable tumors. The Cancer Review 4:129-214 4. Gorer, P. A. 1956. Somerecent work on tumor immunity. Adv. Cancer Res. 4: 149-86 5. Gorer, P. A., Lyman, S., Snell, G. D. 1948. Studies on the genetic and antigenic basis of tumour transplantation. Linkage between a histocompatibility gene and "fused" in mice. Proc. R. Soc. London Set. B 135:499-505 6. Snell, G. D. 1981. Studies in histocompatibility. Science 213:172-78 7. Gross, L. 1943. Intradermal immunization of C3Hmice against a sarcoma that originated in an animal of the same line. Cancer Res. 3:326-33 8. Foley, E. J. 1953. Antigenic properties of methylcholanthrene-induced tumors in mice of the strain of origin. Cancer Res. 13:835-37 9. Baldwin, R. W. 1955. Immunity to methylcholanthrene-induced tumors in inbred rats following atrophy and regression of implanted tumors. Br. J. Cancer 9:652-65 10. Prehn, R, T., Main, J. M. 1957. Immunity to methylcholanthrene-induced sarcomas. J. Natl. Cancer Inst. 18:769-78 l l. Klein, G., Sjogren, H. O., Klein, E.,

Hellstrrm, K. E. 1960. Demonstration of resistance against methylcholanthrene-induced sarcomas in the primary autochthonous host. Cancer Res. 20: 1561-72 12. Old, L. J., Boyse, E. A., Clarke, D. A., Carswell, E. A. 1962. Antigenic properties of chemically-induced tumors. Ann. N.Y. Acad. Sci. 101:80-106 13. Globerson, A., Feldmann, M. 1964. Antigenic specificity of benzo(a) pyreneinduced sarcomas. J. Natl. Cancer Inst. 32:1229-43 14. Bailey, D. W. 1982. Howpure are inbred strains of mice. Immunol. Today 3: 21014 15. Kripke, M. L. 1974. Antigenicity of murine skin tumors induced by ultraviolet light. J. Natl. Cancer Inst. 53: 1333-36 16. Vaage, J. 1968. Nonvirus-associated antigens in virus-induced mouse mammary tumors. Cancer Res. 28:2477-83 17. Morton, D. L., Miller, G. F., Wood, D. A. 1969. Demonstration of tumorspecific immunityagainst antigens unrelated to the mammarytumor virus in spontaneous mammaryadenocarcinomas. J. Natl. Cancer Inst. 42:289-301 18. Carswell, E. A., Wanebo, H. J., Old, L. J., Boyse, E. A. 1970. Immunogenic properties of reticulum cell sarcomas of SJL/J mice. J. Natl. Cancer lnst. 44: 1281-88 19. Koch, S., Zalcberg, J. R., McKenzie,I. F. C. 1984. Description of a murine B lymphoma tumor-specific antigen. J. Immunol. 133:1070-77

Annual Reviews

Annu. Rev. Immunol. 1988.6:465-483. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

UNIQUE TUMOR-SPECIFICANTIGENS

481

20. Hewitt, H. B., Blake, E. R., Walder,A. 1963. Antigenic properties of experS. 1976,A critique of the evidencefor imentalleukemias.I. Serologicalstudies active host defenseagainst cancerbased in vitro with spontaneousand radiationon personal studies of 27 murinetumors inducedleukemias.J. Natl. CancerInst. 31:977-86 of spontaneousorigin. Br. J. Cancer33: 241-59 34. Old, L. J., Stockert, E. 1977. Immuno21. Middle, J. G., Embleton, M. J. 1981. geneticsof cell surfaceantigensof mouse Naturally arising tumorsof the inbred leukemia.Ann. Rev. Genetics 11:127-60 WAB/Not rat strain. II. Immunogenic- 35. Boyse,E. A., Old,L. J., Luell, S. 1963. Antigenic properties of experimental ity of transplanted tumors. J. Natl. leukemias.II. Immunological studies in CancerInst. 67:637-43 22. Urban,J. L., Holland,J. M., Kripke,M. vivo with C57BI/6radiation-induced L., Schreiber, H. 1982. Immunoselection leukemias. J. Natl. CancerInst. 31: 987-95 of tumor cell variants by mice sup- 36. Brown,J. P., Klitzman, J. M., Hellpressedwith ultraviolent radiation. J. strrm, I., Nowinski,R. C., Hellstrfm, K. E. 1978. Antibody response to Exp. Med. 156:1025-41 chemically-inducedtumors. Proc. Natl. 23. Urban,J. L., Kripke, M.L., Schreiber, H. 1986. Stepwiseimmunological selecAcad. Sei. USA75:955-58 tion of antigenic variants during tumor 37. Schlechter, A. L., Stern, D. F., Vaigrowth, J. lmmunol.137:3036-41 dyanathan,L., Decker,S. J., Drebin,J. A., Greene,M.I., Weinberg,R. A. 1984. 24. Bartlett, G. L. 1972.Theeffect of host immunityon the antigenic strength of The neu oncogene: Anerb-B-related primarytumors.J. Natl. CancerInst. 49: gene encodinga 185,000-Mrtumoranti493-504 gen. Nature 312:513-16 25. Koeppen,H., Rowley,D. A., Schreiber, 38. Schlechter, A. L., Hung,M. C., VaiH. 1986. Tumor-specific antigens and dyanathan, L., Weinberg,R. A., Yangimmunological resistance to cancer. In Feng, T. L., Francke, V., Ullrich, A., Conssens, L. 1985. The neu gene: An Mechanisms of HostResistancefor Infectious Aoents,Tumors andAllografts, ed. erb-B-homologousgene distinct from R. M.Steinman, R. J. North, pp. 359and unlinked to the gene encodingthe 86. NewYork:Rockefeller Univ. Press EGFreceptor. Science 229:976-78 26. Koldovsky, P. 1969. Tumor-specific 39. Drebin,J. A., Link, V. C., Weinberg, R. transplantation antigens. RecentResults A., Greene, M. I. 1986. Inhibition of tumor growthby a monoclonalantibody CancerRes. 22:1-74 27. Basombrio,M.A. 1970. Search for comreactive with an oncogene-encoded monantigenicities among25 sarcomas tumorantigen. Proc. Natl. Acad. Sci. induced by methylcholanthrene.Cancer USA 83:9129-33 Res. 30:2458-62 40. Burnet,F. M.1970. A certain symmetry: 28. Basombrio,M. A., Prehn, R. T. 1972. Histocompatibility antigens compared Studiesonthe basis of diversity andtime with immunereceptors. Nature 226: of appearance of chemically-induced 123-26 tumors. Natl. CancerInst. Monogr.35: 41. Coggin, J. H., Anderson,N. G. 1974. Cancer, differentiation and embryonic 117-24 29. Spellman, C. W., Daynes, R. A. 1978. antigens: Somecentral problems. Adv. Ultraviolet light-induced murinesupCancerRes. 19:105-455 pressor lymphocytes dictate specificity 42. Medawar,P. B., Hunt, R. 1983. Can of anti-ultraviolet tumor immune resfetal antigens be used for prophylactic ponses. Cell Immunol.38:25-34 immunization?CIBAFound. Symp. 96: 30. VanWaes,C., Urban,J. L., Rothstein, 160-80 J. L., Ward,P. L., Schreiber, H. 1986. 43. Invernizzi, G., Parmiani, G. 1975. Highlymalignanttumorvariants retain Tumor-associatedtransplantation antitumor-specific antigens recognized by gens of chemically-inducedsarcomata helper T ceils. J. Exp. Med.164: 1547cross-reacting with allogeneic histo65 compatibilityantigens. Nature254:713-14 31. Brent, L., Medawar,P. 1966. Quan- 44. Stauss, H. J., VanWaes,C., Fink, M. titative studies ontissue transplantation A., Starr, B., Schreiber,H.1986.Identiimmunity.VIII. Theeffect of irradiafication of a unique tumor antigen as tion. Proc. R. Soc. LondonB Biol. Sci. rejection antigen by molecularcloning 165:413-23 and gene transfer. J. Exp. Med. 164: 32. Bodmer,W., ed. 1983. Fetal antigens 1516-30 and cancer. CIBAFound.Symp. 96: 1- 45. Pravtcheva, D. D., DeLeo, A. B., 361. London:Pitman Ruddle, F. H., Old, L. J. 1981. Chro33. Old, L. J., Boyse,E. A., Stockert, E. mosome assignment of the tumor-spec-

Annual Reviews

Annu. Rev. Immunol. 1988.6:465-483. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

482

SCHREIBERET AL

ific antigen of a 3-methylcholanthreneinduced mousesarcoma. J. Exp. Med. 154:964-77 46. Roman,J. M., Hirsch, J., Readfiead,C,, Levy, D., DeOgny,L., Dreyer, W. J. 1981.Heritable differences amonggp70like moleculeson C3Hultraviolet lightinduced fibrosarcomas. Transplant. Proc. 13:1782-86 47. Lennox,E. S., Lowe,A. D., Cohn,J., Evan, G. 1981. Specific antigens on methylcholanthrene-inducedtumors of mice. Transplant.Proc. 13:1759-61 48. Ullrich, S. J., Robinson,E. A., Law,L. W., Willingham,M., Appella, E. 1986. A mousetumor-specific transplantation antigen is a heat shock protein. Proc. Natl. Acad. Sci. USA83:3121-25 49. Boyse,E. A. 1970. Cell membrane receptors. In Imrnunosurveillance,ed. R. T. Smith, M. Landy, pp. 5-48. NewYork: Academic 50. Lynch,R. G., Graft, R. J., Sirisinha, S., Simms,E. S., Eisen, H. N. 1972. Myelomaproteins as tumor-specific transplantation antigens. Proc. Natl. Acad. Sci. USA69:1540-44 51. Lampson,L. A., Levy, R. 1979. A role for clonal antigens in cancer diagnosis and therapy. J. Natl. CancerInst. 62: 217-19 52. Miller, R. A., Maloney,D. G., Warnke, R., Levy,R. 1982. Treatmentof B-cell lymphomawith monoclonal anti-idiotype antibody. N. Engl. J. Med. 306: 518-22 53. Allison, J. P., Mclntyre,B., Bloch,D. 1982. Tumor-specificantigen of murine T-cell lymphomadefined with monoclonal antibody.J. lmmunol.129: 22932300 54. O’Neill, H. C., McGrath,M.S., Allison, J. P., Weissman, I. L. 1987.A subset of T cell receptors associated with L3T4 molecules mediates C6VLleukemiacell bindingof its cognateretrovirus. Cell49: 143-51 55. Basombrio,M. A., Prehn, R. T. 1972. Antigenic diversity of tumors chemically-inducedwithinprogenyof a single cell. Int. J. Cancer10:1-8 56. Embleton,M.J., Heidelberger,C. 1972. Antigenicity of mouseprostate transformedin vitro. Int. J. Cancer9:8-18 57. Williams, F. A., Gaynon, J. 1982. Neuronalcell Thy-1glycoprotein: Homology with immunoglobulin. Science 216:696-703 58. Hood,L., Steinmetz, M., Malissen, B. 1983. Genes of the major histocompatibility complexof the mouse.Ann. Rev. Immunol.1:529-68 59. Hood, L., Kronenberg, M., Hunka-

piller, T. 1985.T cell antigenreceptors and the immunoglobulin supergenefamily. Cell 40:225-29 60. Old, L. J. 1981. Cancer immunology: Thesearch for specificity-G.H.AClowes MemorialLecture. CancerRes. 41:361-75 61. Rous, P., Beard, J. W.1935. The progression to carcinomaof virus-induced rabbit papillomas(Shope).J. Exp. Med. 62:523-48 62. Foulds, L. 1954.Theexperimentalstudy of tumorprogression. A review. Cancer Res. 14:327-39 63. Nowell,P. C. 1976.Theclonal evolution of tumorcell populations.Acquiredgenetic lability permitsselectionof variant sublines and underlies tumor progression. Science 194:23-28 64. Farber, E., Camerson, R. 1980. The sequential analysis of cancer development. Adv. CancerRes. 31:125-226 65. Klein, G., Klein, E. 1985. Evolutionof tumors and the impact of molecular oncology. Nature 315:190-95 66. Sukumar, S., Notario, V., Martinzanca, D., Barbacid,M. 1983.Induction of mammarycarcinomas in rats by nitroso-methylurea involves malignant activation of H-ras-1 locus by single point mutations. Nature 306:658-61 67. Muller, R., Slamon,D. J., Tremblay,J. M., Cline, M. J., Verma,I. M. 1982. Differential expressionof cellular ontogenes during pre- and postnatal developmentof the mouse,Nature299:641Y44 68. Weinberg,R. A. 1982. Fewerand fewer oncogenes.Cell 30:3-4 69. Townsend, A. R. M., Skehel, J. J. 1984. The influenza A virus nudeoprotein genecontrols the inductionof both subtype specific andcross-reactivecytotoxic T cells. J. Exp. Med.160:552-63 70. Rajan, T. V. 1987. Is there a role for MHC Class I antigens in the elimination of somatic mutants. ImmunoLToday 8: 171-72 71. Tevethia,S. S., Tevethia,M.J., Lewis, A. J., Reddy, V. B., Weissman,S. M. 1983. Biologyof SimianVirus 40 (SV40) transplantation antigen (TrAg). IX. Analysis of TrAgin mousecells synthesizing truncated SV40 large T antigen. Virology 128:319-30 72. O’Connell,K. A., Gooding,L. R. 1984. Clonedcytotoxic T lymphocytesrecognize cells expressingdiscrete fragments of the SV40tumor antigen. J. ImmunoL 132:953-58 73. Simpson, E. 1987. Non-H-2 histocompatibility antigens: Can they be retroviral products? lmmunol.Today8: 176-78 74. Klein, J. 1978. H2mutations:Their gen-

Annual Reviews

Annu. Rev. Immunol. 1988.6:465-483. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

UNIQUE TUMOR-SPECIFIC ANTIGENS etics and effect on immunefunctions. In Advances in Immunobiology, ed. F. Y. Dixon, H. G. Kunkel. 26:55-141 75. King, W., Patel, M. D., Lobel, L. I., Goff, J. P., Nguyen-Huu, M. C. 1985. Insertion mutagenesis of embryonal carcinomaceils by retroviruses. Science 228: 554--58 76. DeLeo,A. B., Shiku, H., Takahashi, T., John, M., Old, L. J. 1977. Cell surface antigens of chemically-induced sarcomas of the mouse. I. Murine leukemia virus-related antigens and alloantigens on cultured fibroblasts and sarcoma cells: Description of a unique antigen on Balb/c meth A sarcoma. J. Exp. Med. 146:720-34 77. Wortzel, R. D., Philipps, C., Schreiber, H. 1983. Multiplicity of unique tumorspecific antigens expressed on a single malignant cell. Nature 304:165-67 78. Urban, J, L., Schreiber, H. 1987. Hosttumor interactions in immunosurveillance against cancer. Prog. Exp. Tumor Res., Vol. 32 79. Schreiber, H. 1984. Idiotype network interactions in tumor immunity. Adv. Cancer Res. 41:291-42 80. Wortzel, R. D., Urban, J. L., Schreiber, H. 1984. Malignant growth in the normal host after variant selection in vitro with cytolytic T cell lines. Proc. Natl. Acad. Sci. USA 81:2186-90 81. Uyttenhove, C., Maryanski, J., Boon, T. 1983. Escape of mouse mastocytoma P815 after nearly complete rejection is due to antigen loss variants rather than immunosuppression. J. Exp. Med. 157: 1040-52 82. Wortzel, R, D., Philipps, C., Urban, J. L., Fitch, F. W., Schreiber, H. 1983. Independent immunodominant and immunorecessive tumor-specific determinants on a malignant tumor. Antigenic dissection with cytolytic T cell clones. J. Immunol. 130:2461-66 83. Urban, J. L., Van Waes, C., Schreiber, H. 1984. Pecking order among tumorspecific antigens. Eur J. Immunol. 14: 181-87 84. Philipps, C., McMillan, M., Flood, P. M., Murphy,D. B., Forman, J., Lancki, D., Womaek,J. E., Goodenow, R. S., Schreiber, H. 1985. Identification of a unique tumor-specific antigen as a novel class I major histocompatibility molecule. Proc. Natl. Acad. Sci. USA 82: 514044 85. Philipps, C., Stauss, H. J., Wortzel, R. D., Schreiber, H. 1986. A novel MHC Class I molecule as tumor-specific antigen. Correlation between the antibody defined and the CTL-defined target

483

structure. J. Immuno,qenet. 13:93-99 86. Parmiani, G., Carbone, G., Invernizzi, G., Pierotti, M.A., Sensi, M.L., Rogers, M. J., Appella, E. 1979. Alien histocompatibility antigens on tumor cells. Immunogenetics 9:1-23 87. Bortin, M. M., Truitt, R. L., eds. 1980. 1st International Symposiumon Alien Histocompatibility Antigens on Cancer Cells. Transplant. Proc. 12:1-218 88. Bortin, M. M., Truitt, R. L., eds. 1981. 2nd International Symposiumon Alien Histocompatibility Antigens on Cancer Cells. Transplant. Proc. 13:1751-1978 89. Doherty, P. C., Knowles, B. B., Wettstein, P. J. 1984. Immunological surveillance of tumors in the context of major histocompatibility complexrestriction of T cell function. Adv. Cancer Res. 42:1-65 90. Stauss, H. J., Linsk, R., Fischer, A., Watts, S., Banasiak, D., Haberman,A., Clark, I., Forman, J., McMillan, M., Schreiber, H., Goodenow,R. S. 1986. Isolation of the MHCgenes encoding the tumor-specific Class I antigens expressed on a murine fibrosarcoma. J. Immunogenet. 13:101-11 91. Daynes, R. A., Spellman, C. W. 1977. Evidence for the generation of suppressor cells by ultraviolet radiation. Cell. lmmunol. 31:182-87 92. Fisher, M. S., Kripke, M. L. 1977. Systemic alteration induced by mice by ultraviolet light irradiation and its relationship to ultraviolet carcinogenesis. Proc. Natl. Acad. Sci. USA74: 1688-92 93. Kripke, M. L. 1981. Immunologicmechanisms in UVradiation carcinogenesis. Adv. Cancer Res. 34:69-106 94. Hewitt, H. B. 1978. The choice of animal tumors for experimental studies on cancer therapy. Adv. Cancer Res. 27: 149200 95. Rapp, H. J. 1979. Appropriateness of animal models for the immunology of human cancer. Cancer Res. 39: 428586 96. Doll, R, 1980. The epidemiology of cancer. Cancer 45:2475 85 97. Real, F. X., Mattes, M. J., Houghton, A. N., Oettgen, H. F., Lloyd, K. O., Old, L. J. 1984. Class I (unique) tumor antigens on human melanoma. Identification of a 90,000 Dalton cell surface glycoprotein by autologous typing. J. Exp. Med. 160:1219-23 98. Srivastava, P. K., DeLeo, A. B., Old, L. J. 1986. Tumorrejection antigens of chemically induced sarcomas of inbred mice. Proc. Natl. Acad. Sci. USA 83: 3407-11

Annu. Rev. Immunol. 1988.6:465-483. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Annu. Rev. Immunol. 1988.6:465-483. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Annual Reviews www.annualreviews.org/aronline Ann. Rev. Immunol. 1988.6 : 485-512 Copyright © 1988 by Annual Reviews Inc. All rights reserved

MOLECULAR REGULATION OF B LYMPHOCYTE RESPONSE Annu. Rev. Immunol. 1988.6:485-512. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Tadamitsu Kishimoto and Toshio Hirano Institute for Molecularand Cellular Biology, OsakaUniversity, 1-3, Yamada-Oka, Suita City, Osaka565, Japan

INTRODUCTION Antibodiesare one of the key elementsin the immune system. Onthe one hand, they play an essential role in protection against viral and bacterial infections; on the other hand, they are involved in certain autoimmune diseases and in immediate-typehypersensivity. B cells are the only eukaryotic cells that can produceantibodymolecules.Theregulatedproduction of antibody moleculesis one of the mostcomplexexamplesof eukaryotic cell differentiation and one of those most. amenableto scientific investigation. Sincethe discoveryof T andB cell interaction in the antibodyresponse, the mechanism of the regulatoryfunction(s) of T cells in the B cell response has beenone of the central issues in immunology. Theexistence of T cellderived helper factors that promoteB cell proliferation and antibody secretion was recognized in the early and mid 1970s. Dutton and his colleagues (1) as well as Schimpl& Wecker(2) demonstratedthat culture supernatants of murineT cells stimulated by mixedlymphocytereaction or by T cell mitogenscould reconstitute the antibodyresponse of T celldepleted splenic lymphocytes.Kishimotoand his colleagues (3) showed that anti-immunoglobulin (anti-Ig) and T cell-conditioned mediumcould induceIg secretion in rabbit B cells, indicating that twototally antigennonspecificsignals (i.e. cross-linkingIg-receptorsandT cell~terivedhelper factors) could induceIg productionin B cells. Subsequently,the results obtained with rabbit B cells wereconfirmedin the murinesystem. Parker and his colleagues (4) could induce Ig secretion in murineB cells with insolubilized anti-Ig and culture supernatants ofconcanavalinA (ConA)stimulatedT cells. All of these results indicated that the helper function of T cells in antibodyresponsewasmediatedby soluble factors. 485 0732-0582/88/0410-0485502.00

Annual Reviews

Annu. Rev. Immunol. 1988.6:485-512. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

486

KISHIMOTO & HIRANO

Since then, a numberof factors involved in the regulation of B cell response(s) havebeenreported. In a previousreview(5), weproposed the factors for growthand differentiation of B cells be categorizedinto three groups:(i) a factor(s) for the activation of resting B cells, (ii) factor(s) for the growthof activated B cells, and (iii) a factor(s) for final differentiationof activatedB cells into high-rateIg-secretingcells. The application of recombinantDNA methodologyhas allowed the cloning of the cDNAs of factors involved in the regulation of growthand differentiation of B cells. The cDNAs for three distinct factors--BSF1(IL-4) for activation (6, 7), BCGF II (IL-5) for growthof B cells (8), and (IL-6) for Ig-secretion in B cells (9)--have beencloned, andthe involvementof these three moleculesin the process of B cell growthand differentiation has been confirmed. A schemefor B cell differentiation into antibodyproducingcells is depictedin Figure1 on the basis of the function of recombinantBSFs. In this review,wesummarize the molecularstructure, biological function andregulation of expressionof these factors. Since an excellent reviewon BSFI(IL-4) has recently been published (10), in this review we mainly discuss BSF2(IL-6) and the major points of interest on BSF1(IL-4) BCGFII(IL-5). BSF2 (IL-6) BSF2 as a B Cell Differentiation Factor Thepresence of B cell differentiation factor (designated BCDF and subsequently BSF2),whichdoes not have growthactivity and is involved in the final differentiation of B cells to high-rateIg secretion, wasoriginally demonstrated by employinga humanB-lymphoblastoidcell line, CESS, that was responsive to BCDF (11). IgG secretion was induced in CESS BSF-1/IL-4 BCGFII/IL-5

Activation

13SF-2/IL-6

Pro~’~eration Di~erentiation

Figure 1 Schematic presentation of the different phases through which B cells differentiate into plasma cells in response to a variety of interleukins.

Annual Reviews

Annu. Rev. Immunol. 1988.6:485-512. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

BSFs

AND RECEPTORS

487

cells within 48 hr after the addition of PHAconditioning mediumof T cells (PHA-sup).Adsorption of PHA-supwith CESScells removedBCDF activity but not IL-2 activity, indicatingthat CESS cells expressedreceptors for BCDF but not for IL-2, and IL-2 wasnot involvedin the induction of IgG in CESScells. Subsequently, IL-2 and ),-IFN were shown,by use of recombinantformsof these factors, not to be involved in CESSinduction (12). Thepresence of BCDF was confirmedby the establishment of BCDFproducinghumanT hybrid clones (13, 14): culture supernatants of these clones inducedIg secretion in CESScells but lackedother activities, such as BSF1,IL-1, IL-2, and ~-IFN. Therequirementfor at least twodifferent kinds of factors, B cell growth factor (BCGF) and B cell differentiation factor (BCDF),for Ig induction in B cells was demonstrated by the establishment of BCGFand BCDFproducingT hybridomas(13). Stimulation of freshly isolated leukemic cells (B-CLL)with anti-idiotypic antibody (anti-id) and PHA-sup induced monoclonalIgMsecretion (15). In this experimentalsystem, the addition of culture supernatant from BCDF-producing hybridomasdid not induce any Ig secretion or proliferation of anti-id-stimulated B-CLL cells. The culture supernatantof ar~other T hybrid clone could induceproliferation of anti-id-stimulated B-CLLcells, and this activity was designated as BCGF.The addition of BCGFtogether with BCDFcould induce Ig secretion in anti-id-stimulated B-CLLcells, demonstrating that BCDF alone wasnot sufficient and another growthfactor(s) (BCGF) wasrequired for Ig secretion in B cells (12). Asdescribedin the followingsection, the presence of two distinct BCGFs--BCGF I and BCGFII--was subsequently demonstrated.As the BCGF employedin this study could induce the growth of anti-Id-stimulated B-CLLcells, it was called BCGF I. Therefore,the early studies with B-CLL cells indicated that three signals, anti-Ig, BCGF I, and BCDF,inducedgrowthand differentiation of B cells into antibody-secretingcells (12). Internal labeling of newlysynthesized proteins in BCDF-stimulatedCESScells revealed that BCDF augmented the de novosynthesis of secretory-type~ chains of IgG(16). Northernblot analysis and nuclear run-off experimentsdemonstratedthat an increase in ~-chain biosynthesis was due to the induction of mRNA transcription specific for secretory-type heavy chains (16). BCDF also induced increase in mRNA for secretory-type # chains in another B-lymphoblastoid cell line (SKW6 CL-4), suggesting that BCDF does not have isotypespecificity(16, 17). In a subsequent study, an HTLV-l-transformedhumanT-cell line, whichsecreted relatively large amountsof BCDF,was established (18), and the culture supernatants were employedfor the isolation and purification of humanBCDF.With the use of several sequential chro-

Annual Reviews

Annu. Rev. Immunol. 1988.6:485-512. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

488

KISHIMOTO & HIRANO

matographies, BCDFwas purified to homogeneity, and the partial sequenceof the N-terminalaminoacids wasdetermined(19, 20). Since the protein with BCDF activity was purified to homogeneity,this molecule was designated BSF2,following a meeting on nomenclaturein 1983(21). As is described in the following sections, the function of BSF2is not restricted to B cells but showsa widevariety of biological functionson T cells, hematopoieticcells, nerve cells, and hepatocytes.Therefore,it has been proposedthat BSF2be called interleukin 6 (IL-6). Purified BSF2induced IgG or IgMsecretion in B lymphoblastoidcell lines, CESSor SKW6-CL4 cells, respectively, at as little as 3 pM;andthe maximum induction was achieved by approximately 100 pM.On the basis of the N-terminalaminoacid sequence,a synthetic peptide corresponding to the first 13 residues of BSF2was prepared. Anantipeptide antibody raised against the peptide conjugatedto ovalbumincould absorb the BSF2 activity present in PHA-sup,whichconfirmsthat the aminoacid sequence determinedwas actually that of the molecule expressing BSF2activity secreted from PHA-stimulatedlymphocytes(20). Molecular Cloning of the cDNA for BSF2 Purified BSF2was digested with lysylendopeptidase, and nine fragments and the partial aminoacid sequenceof seven fragmentsweredetermined. Onthe basis of aminoacid sequences, synthetic oligonucleotide probes were prepared. The cDNAfor BSF2has been cloned by probing a cDNA library prepared from poly (A)+ RNAof a BSF2-producingT cell line, with groups of synthetic oligonucleotide mixtures (17 bases each). One eDNA clone, designatedpBSF2.38,that specifically hybridizedwith probes was isolated, and the DNA was transfected into COS7cells. The culture supernatant of COS7cells transfected with pBSF2.38DNAinduced IgM secretion in SKW6-CL4 cells, and the BSF2activity generated by COS7 cells could be adsorbed onto and subsequently eluted from an immunoaffinity gel conjugated with the antipeptide antibody described above. Theseresults indicated that pBSF2.38containedthe entire codingregion of BSF2(9). The complete nucleotide sequence of the insert cDNA pBSF2.38and the deducedamino acid sequence are shownin Figure 2a. Because we knowfrom protein sequence data that the sequence of the NHz-terminalregion of mature BSF2is Pro-Val-Pro-Pro..... , it was concluded that BSF2was madeas a precursor consisting of 212 amino acids and processed into a mature form of 184 aminoacids by cleavage betweenthe Ala (-1) and Pro (+ 1) residues. Hydrophobieaminoacids are abundantin residues from -28 to - 1, and this region seemsto be a typical signal peptide that is required for BSF2secretion. The sequenceof BSF2was comparedto other knownproteins, including

Annual Reviews BSFs AND RECEPTORS

Met &sH Set

phe Set

489

Th~ ~er &la Phe Cly Pro Val &la Phe

so

10

30

20

250

Annu. Rev. Immunol. 1988.6:485-512. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

60

70 ]50

120

110

500

C&TTCCTTCTTC~GAJtACCTGTCC&CTGCGC&C JtCAACTT&TGTT~TTCTCT&TGC&G~CT~&~&~G~&~&C&~A 850 900

950 1000

1050

b 6.3 BSF-2 (]L-6)

63,7 191

13.3

54.7

40

164

I

II

38

49

55

aminoacids

114

147

165 bp

AATAAA

36

49

54 aminoacids

G-CSF 47 Ill

IV

162 bp V

Figure2 (a) Nucleotide sequence and deduced amino-acid sequence of the plasmid pBSF2.38 cDNAinsert. Arrow indicates the N-terminus of mature BSF-2; Dots, potential N-glycosylation sites; boxes, presumed poly(A) addition signal sequence. Numbersabove and below the sequence showpositions of aminoacids and nucleotides, respectively (taken from Ref. 9). (b) Comparisonof the gene organization between BSF-2and G-CSF.Boxes represent five exons. The coding and noncoding regions are shownby closed and open boxes, respectively. The numbers above and below the boxes indicate the numbers of amino acids ofexons and those of nucleotides Qf exons for coding region, respectively. The numbers below the lines showthe numbers of nucleotides of introns. The data of the G-CSFgene was based on the report by Nagata et al (23) (taken from Ref. 22).

Annual Reviews

Annu. Rev. Immunol. 1988.6:485-512. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

490

KISHIMOTO & HIRANO

human IL-l~, fl, IFN~, fl, 7, GM-CSF,G-CSF, IL-3, BSF1(IL-4) and BCGFII(IL-5). Only human G-CSFshowed a significant homology with BSF2; the position of four cysteine residues of BSF2match those of GCSF,suggestinga similarity in the tertiary structure of these twomolecules. Moreover, the organization of the genomic gene of BSF2was very similar to that of G-CSF.As shown in Figure 2b, the BSF2gene consists of 5 exons and 4 introns (22). Actually, the two genes have the same number of exons and introns, and the size of each exon is strikingly similar (23). These results suggest that the genes for BSF2and G-CSFmight be evolutionarily derived from a commonancestor gene.

Function of Recombinant BSF2 in the B Cell Responses To express recombinant BSF2(rBSF2) in E. coli, a plasmid, the expression of which is under the control of E. coli trp promotor, was constructed (24). rBSF2was produced as a fusion protein with a part of IL-2; this was further digested with kallikrein, followed by amino-peptidase-P treatment to obtain the mature rBSF2. The rBSF2 was purified by reverse phase HPLC,and the specific activity of rBSF2was 5 × l06 u/rag protein when measured with a B cell line, SKW6-CL4 (25). The addition of rBSF2at concentration between 1 and 10 ng/ml to PWM-stimulated human mononuclear cells augmentedthe production of all classes of Ig. To examine whetherrBSF2in fact acts directly on B cells, purified B cells were stimulated with PWM in the presence of irradiated T cells for 3 days, and Bblast cells were cultured with rBSF2for another 3 days. The result clearly showedthat rBSF2 acted directly on activated B cells to induce Ig production (25a). rBSF2 did not have any growth promoting activity activated B cells. This is in marked contrast with the observations that BSF2functions as myelomagrowth factor and induces the proliferation of myelomacells (26-28). The results obtained with anti-BSF2 antibody indicate that BSF2may be an essential factor for the Ig induction in B cells; in fact, the addition of anti-BSF2 antibody inhibited more than 90% of the PWM-induced IgM and IgG production without affecting the PWM-induced proliferation (25a) (Figure 3). The inhibitory effect was also observed with F(ab’)2 fragment of the antibody. The inhibitory effect of the antibody was seen even if the antibody was added 4 days after initiation in a total 8-day culture. This accords with the findings that BSF2exerts its effect at the final maturation stage of B cells. All of these results obtained with rBSF2 and anti-BSF2antibody indicate that BSF2is essential for the final differentiation of B cells into Ig-secreting cells. Recently, it has been shownthat in vitro growth of plasmacytomasand B cell hybridomas is strictly dependent on a certain factor derived from

Annual Reviews

Annu. Rev. Immunol. 1988.6:485-512. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

BSFs AND RECEPTORS

Concentration

491

(~g/ml)

Fiyure 3 Effects of anti-BSF-2 antibodies on PWM-inducedIg-production of peripheral blood mononuclear cells (PBL). PBL were cultured in the presence of PWM with IgG fraction of rabbit pre-immuneserum (~3, C)) or anti-BSF-2 serum (I, Q). (Left panel) concentrations of IgG (U], I) or IgM(O, Q) in the culture supernatants were measured enzyme-linked immunosorbentassay on day 8. (Right panel) Incorporation of 3H-Thymidine by the cultured cells was determined on day 3.

T cells or monocytes (26-29). Several investigators have observed that successful adaptation of plasmacytomas to primary cultures was only achieved in the presence of feeder cells (30). For some plasmacytomas, feeder cells could be replaced by a soluble factor(s) secreted by macrophages (29, 30, 31), T lymphocytes (26, 32), or a variety of other cells including fibroblasts (28), endothelial cells (33), peripheral blood mononuclear cells (34), and an osteosarcomacell line (MG-63)(27). Stimulation of those cells with several cytokines such as IL-1 or TNFcould induce the production of this factor, which is called hybridomaplasmacytomagrowth factor (HPGF)(27, 28) or interleukin-HPI (26). HumanHPGFwas ified to homogeneity from IL-l-stimulated osteosarcoma cells (MG-63), and a partial NH2-terminal amino acid sequence was determined. The result demonstrated that the partial sequence of HPGFwas identical with that of BSF2, indicating that BSF2has a growth-promoting activity for plasmacytomas (27). A BSF2-dependent hybridoma line has been established (25a). The growth of a BSF2-dependent hybridoma line (MH60.BSF2)was induced by rBSF2 in a dose-dependent fashion, and the maximumresponse was achieved at the concentration of 20 pg/ml, which is approximately 100fold less than the level required for Ig-induction in B cell lines. A similar observation was reported by Van Snick et al (26)--that plasmacytomas required for their half-maximal growth a concentration of 30 pM, which

Annual Reviews

Annu. Rev. Immunol. 1988.6:485-512. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

492

KISHIMOTO & HIRANO

is 200 times higher than that required by hybridomacells. The reason hybridomacells are more sensitive to BSF2than are plasmacytomasor B cell lines is not known.The observation that humanBSF2induces proliferation of murinehybridoma cells suggests a similarity of the molecular structure of humanand murine BSF2. Recently, the partial aminoacid sequenceof murineHPGFwas determined (35) and found to be completely different from that of human BSF2.Severalpossibilities shouldbe considered:(i) a portion of molecule importantfor its function maybe conserved,or (ii) two different kinds BSF2are present, similar to IL-1 ~ and/~ or TGF~ and Theintraperitoneal injection of mineral oil is well knownto generate plasmacytomasin mice (36) and to provide a good condition for the expansion of hybridomacells. This phenomenon can be explained by the fact that granulomasgenerated by mineral oil produce BSF2which is essential for the growthof plasmacytoma cells. Actually, it wasshownthat adherentperitoneal cells inducedby mineral oil produced50-fold greater amountsof growth factor activity on a plasmacytomacell line (29). Therefore, deregulation of BSF2expression mightbe one of the elements in the pathogenesis of plasmacytomas.This might be applied to human multiple myelomas.Preliminarystudies demonstratedthat freshly isolated myelomacells producedBSF2and expressed BSF2receptors. Moreover, rBSF2augmentedthe in vitro growth of myelomacells, and anti-BSF2 antibody inhibited the in vitro growthmyeloma cells (M. Kawano and T. Hirano,unpublisheddata). Theseresults indicate that BSF2is an autocrine growth factor for humanmyeloma. Pleiotropic

Functions of BSF2

Severalinterleukins are knownto havea widevariety of biological activities. The molecular cloning of cDNAscould showthat the biological factors originally detected on the basis of individual activities havethe same structure. The samewas also true for BSF2.The cDNAs for 26-kd protein (41) and IFNfi2(42) were recently cloned, and the results showed that 26-kd protein and IFN/~2are identical to BSF2. The26-kdprotein (or IFN/~2)wasoriginally describedas the translation product of a mRNA whosesynthesis occurs in humanfibroblastoid cells in response to various stimuli, such as IL-1, TNF,PDGF and poly (I) poly (C), someof whichalso induce the productionof classical IFN~(3740). Weissenbach et al (37) reportedantiviral activity in their IFN/~2,which could be neutralized by anti-IFN/~, although its IFNactivity wasonly 1% to 2%that of IFN/~. Onthe other hand, Contentet al (38) did not find any antiviral activity in their 26-kdprotein. Theresults suggestthat BSF2 mayhavea widevariety of biological functions, andits effects maynot be

Annual Reviews

Annu. Rev. Immunol. 1988.6:485-512. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

BSFs AND RECEPTORS

493

restricted to B cells andto plasmacytomas. Oneof the importantissues is whetherBSF2(26-kd protein/IFNfl2) has any antiviral activity. Byutilizing rBSF2, anti-BSF2, and anti-IFNfl antibodies, it was shownthat rBSF2did not possess antiviral activity, and that BSF2and IFNfl were not antigenically related (42a). Alack of IFNactivity in recombinant26kd protein expressedin Xenopusoocytes wasalso reported (43). In fact, these results exclude BSF2from the IFN family. By employingrecombinant molecules, Poupart et al demonstratedthat the major function of the 26-kd protein wasthe induction of growthand differentiation of B cells (43). However,the function of BSF2is not restricted to B cells; it exerts a stimulatoryeffect on hepatocytes(44, 45), nervecells (T. Sato, T. Hirano, T. Kishimoto,Y. Kaziro, in preparation), and hematopoieticcells. The acute phase response is a systemic reaction to inflammationor tissue injury, characterized by significant changesin the concentrationof many hepatically derived plasmaproteins knownas acute-phasereactants (46). Withthe use of in vitro hepatocyte cell cultures, IL-1 was shownto be involved in acute-phase response (47). However,recent observations suggest that the full hepatic acute-phase protein response cannot be explainedby the action of IL-1 alone (48, 49). Byusing recombinantBSF2, Gauldie et al (44) and Anduset al (45) demonstratedthat BSF2induced the synthesis, of all the majoracute phaseproteins in humanor rat hepatomacells. Moreover,the activity of hepatocyte-stimulating factor present in conditioned mediumfrom humanmonocyteswas neutralized by antiBSF2(45). Theresults clearly showedthat BSF2functions as a hepatocytestimulating factor in inducingacute-phaseprotein response. IL- 1 stimulationof glioblastomacells or astrogliomacells wasfoundto induce the expressionof BSF2specific mRNA (22), and this suggests that BSF2mayhavecertain effects on nerve cells. It is well knownthat nerve growthfactor (NGF)induces the phenotypicshift in chromaffincells and in their neoplastic counterpart,the clonal cell line PC12,whichresults in their neuronaldifferentiation accompanied by chemical, ultrastructural, and morphological changes (50). Incubation of PC12cells with BSF2 inducedthe typical differentiation of those cells into neuronalcells. The differentiation inducedwith BSF2wassimilar to that observedwith NGF, although BSF2and NGFuse completelydifferent receptors expressed on PC12 cells. Anotherexampleof a cytokine with potent effects on both neuronsand lymphocytes is neuroleukin(51, 52). This factor wasinitially purified from mousesalivary gland, and cDNA encoding the factor has been cloned. Thesequenceencodesa protein of 558 aminoacids with thre’e potential N-linkedglycosylation sites. Nostrong homology to any of the sequences

Annual Reviews

Annu. Rev. Immunol. 1988.6:485-512. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

494

KISHIMOTO & HIRANO

of knownpolypeptide growthfactors or cytokines wasfound. It showsa partial sequence homologywith the envelope protein of HIV(gp 120). Recombinantneuroleukin supports the continued survival in culture of spinal and sensory neurons. Neuroleukinis producednot only by salivary gland but also by several tissues includingskeletal muscleand brain. It is interesting that neuroleukinis a secretory product of lectin-stimulated T cells and induces immunoglobulin synthesis by cultured humanperipheral mononuclear cells. Neuroleukinmayact directly on B cells, but the presence of T cells and monocytesis essential for neuroleukin-inducedIgsecretion in B cells. Antineuroleukininhibits PWM-induced Ig-production, indicating that neuroleukin is one of the essential factors for the Iginduction. Suppression of PWM-induced Ig-secretion was obtained only whenantineuroleukin monoclonalantibody was added early (day 0). However, the addition on day 3 or later did not inhibit subsequent Igproduction. In contrast, the addition of anti-BSF2antibody even on day 4 could inhibit PWM-induced Ig production. The results suggest that in addition to IL-4 and IL-5 an early event that triggers activation or clonal expansionof B cells is neuroleukin-dependent, while the final maturation stage of activated B cells is neuroleukin-independent but BSF2-dependent. As described, BSF2is producedin various tissues by various stimuli, i.e. in T cells by mitogenor antigen stimulation in the presenceof monocytes or IL-I; in fibroblasts by IL-1, TNF,PDGF or poly (I): poly (C); glioblastomasby IL-1; and in monocytesby LPSor other activators. BSF2 showsa wide variety of biological functions not only in the antibody productionin B cells, but also in the generationof plasmacytomas, in the differentiation of neurons, in the induction of acute-phase proteins in hepatocytes, and in the activation of hematopoieticstem cells at the Go phase into the IL-3 dependentstage. Figure 4 summarizingthe functions of BSF2indicates that BSF2mayplay an important role in the hostdefense mechanismthrough the activation of B cells, nerve cells, and hepatocytesand hematopoieticcells. BSF1 (IL~4)

AND BCGFII (IL-5)

BSF1 as an Activation

Factor

BSF1(IL-4) wasinitially describedon the basis of its ability to induce DNA synthesis in B cells stimulated with anti-IgM. This activity was identified in the culture supernatantsof a murineT-cell line, EL-4(53), of a humanT-hybridoma cell line (13), andwasinitially designatedB-cell growthfactor 1 (BCGF1),since the presence of two distinct factors with BCGFactivity has been demonstrated, namely BCGFIand BCGFII. Subsequently, BCGFIwas designated BSF1on the basis of discussion at

Annual Reviews BSFs

AND RECEPTORS

495

B cells Ig-production hyb~idoma /plasmacytoma ~’owth T cells IL-2 receptorinduction CTLinduction BSF-2/IL-6

Hepatocytes acutephase proteinproduction

Annu. Rev. Immunol. 1988.6:485-512. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Hematopoietic cells multi-CSFfunction

fibroblasts astrocytes

Fiyure 4 Schematic presentation

Nervecells induction of differentiation Noantiviral activity

of the pleiotropic

activities

of BSF-2,

the nomenclaturemeeting(21). Extensive studies on the molecularand immunological characterization of BSF1 havebeencarried out (10). BSF has been purified to homogeneity,and a rnonoclonal antibody was prepared against BSF1(54). BSF1has a molecular weight of 20 kd, based on data from sodiumdodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis; and approximately10 pg/ml of BSF1is sufficient to induceproliferation of anti-IgM-stimulated B cells (55). Initially, BSF1wasthought to be a "growthfactor" analogousto IL-2 whichacts in late G1phaseto stimulate the activated cells to synthesize DNA.However,several experimentalresults indicated that BSF1acts on resting B cells in the absenceof any recognizedcostimulant:(i) resting cells cultured with BSF1undergoa small but significant increase in cell volumeandan improved viability (56); (ii) resting B cells treated with showa striking increase in the density of class II majorhistocompatibility complex(MHC) molecules(57); and (iii) pretreatment of resting B for 24 hr with BSF1is found to speed up their subsequentresponse to anti-IgMand BSF1by about 12 hr (58). All of these results indicate that BSFIexerts its action on resting B cells and is not a growthfactor but rather an "activation factor" or "competencefactor." In certain experimentalsituations, BSFIfunctions as a differentiation factor of B lymphocytes. In fact, murine B cells treated with lipopolysaccharide(LPS)will secrete IgMand IgG3but little or not IgGvThe addition of culture supernatants from several stimulated T ceil lines or hybridomascauses the secretion of IgG~and results in the partial sup,pression of IgG3production. Thefactor mediatingthis activity wasoriginally designatedB cell differentiation factor for IgG~(BCDFy) (59).

Annual Reviews

Annu. Rev. Immunol. 1988.6:485-512. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

496

KISHIMOTO & HIRANO

biochemicalcharacterization of BCDF~ indicated that it was similar to BSF1in both its molecularweight and its isoelectric point (60). Subsequently, a monoclonalanti-BSF1 antibody was shownto inhibit the activity of BCDF7 in the culture supernatants, confirmingthat BSF1and BCDF7 are the same molecule.(61). Nomaet al (6) cloned the eDNA BSF1from a cDNAlibrary derived from a BCDF~-secretingmurine T cell line designated2.19; this wasdone by monitoringthe ability of the expressed product to induce LPSblasts to secrete IgG~. The study confirmed that BSF1indeed has IgG~-inducingactivity. Simultaneously,Lee et al (7) cloned the cDNA for BSFIby selecting the cDNA encoding the activity of mastcell growthfactor-2 (MCGF-2) and T cell growthfactor2 (TCGF-2) (the latter is distinct fromIL-2). Theyshowedthat the culture supernatant of Cos7cells transfected with this cDNA not only had MCGF and TCGF activity but also had the capacity to induce the proliferation of anti-IgM-stimulatedB cells as well as to induce class II MHC molecules on resting B cells. Together these results obtained from cDNA cloning studies demonstratedthat BSF1has a widevariety of functions: (i) BCGF activity with anti-IgM,(ii) Ia (class II MHC)-inducing activity, (iii) inducing activity, (iv) MCGF activity, and (v) TCGF activity. Since function of BSF1is not restricted to B ceils, it is proposedthat it be designated IL-4. With regards to MCGF activity of BSF1,more detailed study was done by Hamaguchi et al (62). They showedthat BSF1and IL3 exerteda synergistic effect on the proliferation of connective-tissuetype mast cells. On the other hand, IL-3 alone could induce the maximum proliferation and differentiation of mucosalmast cells frombone marrow precursors. Subsequently, a cDNA for humanBSF1(iL-4) was also cloned, based on homologywith a mouseBSF1cDNA;the expressed product could induceproliferation of anti-IgM-stimulated B cells, as well as T cells and mastcells (63). Kikutani et al (64) recently cloned the cDNA for human lymphocytereceptors for IgE (FceRII), and they demonstratedthat BSF inducedthe expressionof FceRII on B cells but not. on T cells. Aswill be described later, BSF1is responsible for the induction of IgE as BCDFe. All these results including (i) MCGF activity, (ii) FceRII induction, (iii) BCDFe activity indicate that BSF1is intimately related to immediate type hypersensitivity. BCGFH as a Growth and Differentiation Factor BCGF activity distinct fromBSF1 wasinitially identified in an alloreactive humanT-ceil clone (d4) (65) and a humanT-hybridclone (13). Theactivity wasseparated from BSF1activity (which was initially designated 20 KBCGF or BCGF-I)by gel filtration, and it had an apparent weight of 50

Annual Reviews

Annu. Rev. Immunol. 1988.6:485-512. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

BSFs

AND RECEPTORS

497

kd. The 50 K-BCGFshowed a synergistic effect with 20 K-BCGFin the induction of proliferation of anti-IgM-stimulated B cells (13, 65). In murine T cells, Swain & Dutton reported a similar BCGFsecreted from a longterm alloreactive murine T cell line (66). The activity of (DL) BCGF could be assayed by its ability to promote the proliferation of dextran sulfate (DXS)-stimulated murine B cells or the BCLI leukemic cell line. The activity waseluted in the fraction with a weightof 50-70kd on gel filtration. Since 20 K-BCGFor BCGF-I(BSF1) did not promote the proliferation of DXS-stimulatedB cells or the BCL~ leukemic cell line, it appeared that (DL) BCGFand human 50-kd BCGFwere molecules distinct from 20 BCGF(BSFI). On the basis of these studies, Swain et al (67) designated 50 K-BCGFas BCGF-II. Subsequent studies carried out by Swain and her colleagues with murine BCGFII and by our group with human BCGFII have shown that BCGFII could induce not only proliferation but also Ig secretion in DXS-stimulated murineB cells or in the BCL~leukemic cells (18, 66, 67). The activities induce proliferation and Ig secretion were copurified by several fractionation procedures: this implies that BCGFIIhas both activities. Similar factors with both growth and differentiation activities have been reported by several investigators; with the use of a single-cell assay for B-cell responsiveness, Pike et al (68) reported a murineB cell growthand differentiation factor (BCDF)secreted from EL-4 cells. Takatsu et al (69) extensively studied the physicochemical and immunologicalproperties of the so-called "T151-TRF." T151-TRF secreted from a murine T hybridoma showed both growth and differentiation activities. In fact, it could induce proliferation of DXS-stimulatedmurine B cells as well as did the BCL~cell line. The same purified preparation induced IgMsecretion in the BCL~ cell line and in antigen-primed B cells, which suggests that B151-TRF belongs to the category of BCGFII. Recently, Kinashi et al (8) cloned the cDNAfor BCGFIIactivity from the 2.19 T cell line and confirmed that recombinant BCGFIIhas both growthand differentiation activities on activated B cells as well as on the BCL~leukemic cells. Furthermore, the recombinant BCGFIIwas found to act not only on B cells but also on T cells and on eosinophils, to induce cytotoxic T cells together with IL-2 (70) or to promote the maturation the latter (71). Harada et al (72) prepared a monoclonalantibody against purified T 151-TRFand demonstrated that the antibody could absorb all the activities of BCGFII,TRF,and eosinophil differentiation factor. In contrast to BCDF7and BCDFeactivities of BSF1, BCGFIIcould function as BCDF~and preferentially induce IgA production (73). Since BCGFII showedseveral different activities not only on B cells but also on T cells and eosinophils, they proposed to designate BCGFII/TRFas IL-5. By gel

Annual Reviews

Annu. Rev. Immunol. 1988.6:485-512. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

498

KISHIMOTO & HIRANO

filtration, BCGFIIactivity was found in the fraction with a molecular weight of 50 K. However, the deduced amino acid sequence of the cloned BCGFII/TRF demonstrated that BCGFII/TRFconsisted of 133 amino acids including a signal peptide of 21 amino acids (8). Therefore, BCGFII in culture supernatants of T-cell lines maybe present in a dimer form. Azuma et al (74) cloned the cDNAfor human BCGFII/TRF based homology with a murine BCGFII cDNA. Although human BCGFII/TRF could also induce Ig secretion in activated B cells in a way similar to BSF2, the cDNAsequences do not display any similarity between the two molecules. As the studies with human recombinant BCGFII(IL-5) are still limited, it is not knownwhetherthis factor plays a central role in the regulation of the growth of human B cells. The relationship between BCGFII(IL-5) and high and low molecular weight BCGFsis also not known. The molecular cloning of the cDNAfor low molecular weight BCGFwas reported, and this was different from BCGFII(IL-5) on the molecular basis (74a). However, nothing is knownabout the biological function of the expressed product of low molecular weight BCGF.Further studies will be required for the molecules involved in the regulation of the growth of humanB cells.

RECEPTORFOR BSFs Noneof the receptor molecules for the various interleukins except one of two chains of IL-2 receptors (Tac antigen) (75, 76) has been characterized. The reason may be the low number of receptor molecules on target cells. The receptors for BSF2or BSF1are not the exception, but the purified or recombinant BSFmolecules have made it possible to detect and characterize the homologousreceptors.

BSF2Receptors BSF2receptors were studied by using radioiodinated recombinant BSF2 (25). There was a single class of receptors with high affinity (kd = 3.4 x 10-l° M) on a BSF2- responsive B lymphoblastoid cell line, CESS; and the number of receptors was 2700 per cell. Binding of ~25IBSF2to CESSwas competitively inhibited by unlabeled BSF2but not by IL-1, IL-2, IFNfl, IFN7 and G-CSF;this indicates the presence of BSF2specific receptors. As suggested by a wide variety of biological functions of BSF2,its receptors are distributed on various cell types. The largest number of receptors (more than 104 per cell) could be detected on humanmyelomacell line, U266, and thus fits the function of BSF2as plasmacytomagrowth factor. With regards to the B lineage cells, all EBV (Epstein-Barr virus) transformed B lymphoblastoid cell lines expressed

Annual Reviews

Annu. Rev. Immunol. 1988.6:485-512. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

BSFs

AND RECEPTORS

499

BSF2receptors, whereasnone of the Burkitt’s lines expressed the receptors. Several other cell lines--the histiocytic line (U937), the promyelocyticline (HL60), the astrocytoma line (U373), and the glioblastoma line (SK-MG4), in which BSF2was inducible with IL-1 or TPA(22)--displayed BSF2 receptors, which suggest an autocrine mechanismin BSF2function. The hepatocyte cell line (Huh-6) also expressed receptors, as might be expected from the function of BSF2as hepatocyte stimulating factor (44, 45). BSF2receptors were not present on normal resting B cells but were expressed on activated B cells with a kd of 3-5 × 10-1° M, in contrast to the expression of BSF1receptor on resting B cells (77). The results fit the functions of BSF2and BSF1; BSF2acts on B cells at the terminal differentiation stage to induce immunoglobulin production, while BSF1 acts mainlyon resting B cells for activation. In contrast to B cells, resting T cells expressed BSF2receptors, the dissociation constant and number of which were comparable to those on B cells. The result suggests the possible involvementof BSF2in the early activation of resting T cells. In fact, the induction of IL-2 receptors on T cell lines by BSF2was demonstrated (78). The molecular structure of the BSF2receptor is not known. A preliminary study with cross-linking reagents demonstrated the involvement of two peptide chains in BSF2receptors. A two-chain model has been shown for IL-2 receptors--a 45-kd ~ chain (Tac molecule) and 75-kd/~ chain (79, 80). Similar organization with two peptide chains maybe posited for the receptors of IL-3 and NGF(81). Therefore, the BSF2receptors may have a similar organization. BSF1 Receptors As described, BSF1also shows a wide variety of biological functions on B cells, T cells, and several other hematopoietic cells (10). As expected their function, the murine BSF1receptors are widely distributed to the hematopoietic-lineagecells, such as B cells, T cells, macrophages,and mast cells (77, 82, 83). The numberof receptor molecules on resting B or T cells was estimated to be a few hundred per cell, and the activation of B cells with LPSor anti-IgM increases the receptor number to about a thousand (77, 82). The receptor was homogeneouswith regard to its affinity with kd of 3 × 10-11 M, and no low-affinity receptors were detected. Relatively higher numbersof receptor molecules (~ 3000) were detected on mast cell lines (77). Xid B cells from CBA/N mice are not responsive to soluble antiIg and BSF1in proliferation (84). However,the BSF1receptors on Xid cells were comparable to those on normal B cells in their number and affinity (83). Monoclonalantibodies against murine B cell differentiation antigens, Lyb2and LFA1, could showan agonistic effect on the activation

Annual Reviews 500

KISHIMOTO & HIRANO

Annu. Rev. Immunol. 1988.6:485-512. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

ofmurineresting B cells (85, 86). However,these antibodies did not inhibit the binding of BSF1to B cells (77), indicating that neither Lyb2nor LFA1 is the receptor for BSF1.Cross-linking reagents madeit possible to detect a new species weighing about 75 or 80 kd on SDS-PAGE analysis; this suggests that the BSF1receptor may be composedof a single chain and may weigh approximately 55 or 60 kd (77, 82). At present, nothing known about human BSF1 receptors. However, the situation may be similar to that of the murine receptors, since humanBSF1also showed a wide variety of functions (63). Signals

Through

BSF Receptors

Several studies have shown that the activation of protein kinase C is involved in the transduction of the signal(s) through antigen receptors B and T cells (87, 88); (i) anti-Ig induces increased incorporation 32p into phosphatidylinositol (89); (ii) anti-Ig induces the translocation protein kinase C to the membrane(87); and (iii) anti-Ig induces rapid increase in intracellular Ca+ + levels (90). Unlike anti-Ig stimulation, BSF1 2+ binding to its receptor does not induce phosphoinositol metabolism, Ca mobilization, protein kinase C translocation, or membranedepolarization (91). The only detectable biochemical change was the phosphorylation a 42-kd protein upon stimulation of isolated B cell membranesin the presence of 32p-ATP(92). Therefore, at the momentlittle is knownabout the biochemical mechanismof the signal transduction provided by BSF1. One of the interesting findings with regard to the BSF1signaling was that ~-IFN inhibits the BSFl-induced activation of B cells, although ~-IFN does not block the binding of BSF1to its receptors (93). With regard to the signaling by BSF2, the situation is very similar to that by BSF1. In a B lymphoblastoid cell line, where BSF2induced an increase in the transcription of mRNA for secretory-type heavy chains of Ig genes, no phosphoinositol (PI) metabolism, 2÷ mobilization, pr otein kinase C translocation, membrane depolarization, or protein phosphorylation was observed (H. Kishi and T. Kishimoto, unpublished observations). In the same B-lymphoblastoid cell line, anti-Ig-stimulation induced PI metabolism, an increase in intracellular Ca:÷, and protein kinase C translocation. However, BSF2did not augment or suppress the anti-Ig-induced PI metabolism and Ca2+ mobilization. Furthermore, antiIg did not affect the BSF2-inducedIg secretion in a B cell line. As indicated, BSF2 functions as a potent growth factor for plasmacytomas or hybridomas. Receptors for growth factors such as EGF, PDGF,insulin, and M-CSFhave a domain of tyrosine kinase in their cytoplasmic portion (94). The involvementof tyrosine kinase in the signal transduction of BSF2 in plasmacytoma may be supposed. One of the

Annual Reviews BSFs AND RECEPTORS

501

interesting findings with BSF2wasthat the concentrationof BSF2required for the maximum induction of growthof BSF2-dependent hybridomacells was100-foldless than that for Ig inductionin B cells (25a, 26) as described in the section on the function of recombinantBSF2in the B cell response. Therelationships betweengrowthanddifferentiation signals, affinity, and the molecularorganization of BSF2receptors are the subject for future studies.

Annu. Rev. Immunol. 1988.6:485-512. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

BSFs

AND IMMUNOLOGICAL

DISORDERS

BSF2 and Autoimmune Diseases BSF2maybe a principal lymphokinefor generalized autoimmune diseases. As described, BSF2can be expressed not only in lymphocytesbut also in several different tissues (22). Severaltumorcells such as cardiac myxoma, cervical cancercells, and bladder-cell carcinomasalso aberrantly produce large amountsof BSF2,and patients with such tumors often showautoantibody production and autoimmunesymptoms(20). Oneof the typical examplesis cardiac myxoma,whichis a benign intraatrial heart tumor. Approximately 30%of the patients with cardiac myxomashow autoimmunephenomena and autoantibody production (95). Theculture supernatant of cardiac myxoma cells wasfound to display a high BSF2activity, whichcould be absorbedwith antipeptide antibody prepared on the basis of the N-terminalaminoacid sequenceof BSF2(20). Northernblot analysis with the cDNA for BSF2confirmed the elevated expression of BSF2 mRNA in cardiac myxoma cells (9). A similar situation was found in patient with cervical cancer, whoshowedSj6gren-like syndromeand high titers of autoantibodies in the serum. The tumor cells secreted large amountsof BSF2moleculeswhichcould be adsorbedwith the antipeptide antibody (20). The patient’s autoimmune symptomsdisappeared 3 months after the surgical removalof the tumor.All these results strongly suggest that the overproductionof BSF2maybe responsible for the autoantibody production. The most striking exampleof the abnormal production of BSF2and autoimmune diseases is rheumatoidarthritis (RA).Previously, A1-Balaghi et al (96) demonstratedBCDF activity in synovial fluids from patients with active RA;this activity inducesIg secretion in activated B cells or B cell lines. RIAassay with anti-BSF2antibody indicated high levels of BSF2in synovialfluids of the affected joints in 22 out of 25 active cases of RA(T. Hirano, submitted), whereasBSF2was not detectable in most synovial fluids from patients with active osteoarthritis. Anincreased expression of BSF2mRNA in cells isolated from synovial fluids of RA patients wasalso observedwith the cDNA for BSF2.It is noteworthythat

Annual Reviews

Annu. Rev. Immunol. 1988.6:485-512. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

502

KISHIMOTO & HIRANO

IL-1 wasnot detected in mostsynovial fluids of RApatients. Theseresults strongly suggest that unregulated expression of the BSF2gene maybe involved in the pathogenesisof RA. Anotherexampleof a disease related to the possible deregulation of BSF2production is Castleman’sdisease, whichis characterized by lymphoadenopathywith massive infiltration of plasma cells and systemic symptoms such as hyper-~-globulinemia,increase in acute phase proteins, and anemia (97). In some advanced cases, monoclonalgammopathy and plasmacytomasare observed. The study done by K. Yoshizaki and T. Kishimoto (manuscript in preparation) demonstrated abnormal production of BSF2by the affected lymphnodes,suggestingthat BSF2induces massiveproliferation of plasmacells. All these results indicate that unregulated expression of BSF2mRNA is involved in the pathogenesis of RAand Castleman’sdiseases. As described in the section on BSF2,BSF2 is producedby varioustissues with variousdifferent stimuli, whichsuggests the presence of a unique regulatory mechanism in the expression of BSF2. It wasdemonstrated that there are several different transcription initiation sites in the BSF2gene, anddifferent sites maybe used for the expression of the BSF2gene in different tissues (22). The study on the molecular mechanism regulating the expressionof the BSF2gene will elucidate some of the events in the complexpathogenesisof systemicautoimmune diseases. BSF1 and Immediate- Type Hypersensitivity BSF1 has an interesting role in immediatetype hypersensitivity. It induces IgEsecretion in LPSblasts: in in vitro cultures stimulatedwith LPS,the addition of BSF1augmentsIgE secretion morethan 100-fold (98). The vivo effect of BSF1on IgE production was also substantiated (99). Mice infected with the larvae of the helminthNippostrongylus brasiliensis display approximately a 100-fold increase in serum IgE concentration. When inoculated with ascitic fluid containing monoclonalanti-BSF1antibody, these mice have a diminishedincrease in serumIgE, whichindicates the role of BSF1in IgE induction in vivo. Mosmann et al (100) demonstrated that T cells producingBSF1were distinct from those for IL-2 or ~-IFN: Theyexaminedseveral T-cell clones for lymphokineproduction and found that T-cell clones producing BSF1never expressed IL-2 or 7-IFN. They designatedT cells for BSF1production as TH2and T c~lls for IL-2 or 7IFN as TH1. In the early 1970s, Kishimoto & Ishizaka (101) proposed that helper T cells involved in the induction of the IgE response were different from those for the IgG response. Moreover,they demonstrated that the soluble helper factor for IgEwasdifferent fromthat for IgG(102). Now,those previous studies can be interpreted on the molecular basis:

Annual Reviews

Annu. Rev. Immunol. 1988.6:485-512. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

BSFs

AND RECEPTORS

503

TH2cells secrete BSF1,whichhelps the induction of IgE; and TH1cells secrete y-IFNand IL-2, whichhelps the induction of IgG(103). Several studies have demonstratedthat IgE binding factors negatively regulate the IgE antibodyresponse(104, 105). It is reasonableto assume that the IgE binding factor(s) is the soluble fragmentsof lymphocyteFc~ receptors (FceRII). It is interesting to note that BSF1augmentsthe expressionof FceRII on B cells at the level of transcription (64). Atopic patients with high levels of serumIgE also showelevated levels of serum FceRII (M. Suemura,E. Barsumian, H. Kikutani, T. Kishimoto, submitted). Therefore, in atopic patients, overproduction of BSF1maybe responsible for the elevated levels of IgE and FceRII. Moreover,BSF1 wasfoundto inducethe proliferation anddifferentiation of mastcells (7, 62). These results suggest that BSF1maybe the principal lymphokine for immediatetype hypersensitivity, and abnormalregulation of BSF1 productionmaybe present in atopic patients. In contrast to BSF1,y-IFN stimulates the expression of the IgG2a isotype and inhibits the production of IgE (103). y-IFNhas also been shownto enhance the expression of humanFc~ receptors analogous to mouseFcRIspecific for lgG2a(106). Thesefindings suggest that ),-IFN maybe important in immuneresponse(s) in whichADCC,opsonization, and complement-mediated lysis play importantprotective roles. As ),-IFN is shownto be producedby TH1cells (100), TH1helper cells may preferentially activatedfor viral and bacterial infection, while TH2helper cells play a role in parasite infection andimmediated type hypersensitivity through BSF1production. CONCLUSION

AND PERSPECTIVES

Themolecularstructures of BSFswhichmediatethe regulatory functions of helper T cells in the antibody response have been determinedby the cloning of their cDNAs.As summarized in Table 1, BSFsare divided into three categories with respect to their activity: (i) a factor mainlyfor the activation of resting B cells (BSF1/IL-4),(ii) a factor for the growth of activated B cells (BCGF-II/IL-5),and (iii) a factor for the terminal differentiation of B cells into Ig-secreting cells (BSF2/IL-6).However, the studies with recombinantBSFsdemonstratedthat BSF1/IL-4 and BCGFII/IL-5 can also function as differentiation factor(s) for the induction IgG~ and IgE or IgA, respectively, and BSF2/IL-6is a potent growth factor for plasmacytomacells. Moreover,the function of BSFsis not restricted to B cells, althoughthey can showpleiotropie activities on a wide variety of cells; for example,BSF2/IL-6stimulates hepatocytesto induce acute phase proteins. It also acts on PC12cells to induce their

Annu. Rev. Immunol. 1988.6:485-512. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Annual Reviews 504 KISHIMOTO & HIRANO

Annu. Rev. Immunol. 1988.6:485-512. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Annual Reviews BSFs AND RECEPTORS 505

Annual Reviews

Annu. Rev. Immunol. 1988.6:485-512. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

506

KISHIMOTO& HIRANO

differentiation into neuroncells. Asexpectedfromtheir pleiotropic functions, receptors for BSFsare widelydistributed on various cell types. The interactions of BSFswith their receptors do not generate any of the known biochemicalresponses. Molecularcharacterization of the receptors will be essential for the elucidation of the mechanisms of the signal transduction and the expression of the BSFfunctions. The involvement of the unregulated production of BSFsin several immune disorders is suggested, i.e. BSF1/IL-4 and immediate-typehypersensitivity, and BSF2/IL-6and autoimmunediseases, especially rheumatoid arthritis and myelomas.Future studies on the molecular mechanisms of the normaland abnormalregulation of the expression of the BSFs-genes will provide essential informationconcerningthe pathogenesis of these diseases. ACKNOWLEDGMENTS

The studies described here were supported by a Grant-in-Aid for the special project research from the Ministry of Education, Science, and Culture. Theauthors wouldlike to thank our scientific colleagues who aidedin the preparationof this reviewby providingpreprints or by allowing us to quote from their unpublishedwork. Wealso wish to thank Dr. EdwardBarsumianfor his critical review of the manuscriptand Mrs. J. Shibuyafor her expert editorial assistance.

Literature Cited 1. Dutton, R. W., Falkoff, R., Hirst, J. dent soluble factor. J. Immunol.123: A., Hoffman,M., Kappler, J. W,Kett931-41 man,J. R., Lesley,J. R., Vann,P. 1971. 5. Kishimoto,T. 1985. Factors affecting Is there evidence for a non-antigen B-cell growthand differentiation. Ann. specific diffusable chemicalmediator Rev. Immunol.3:133-57 for the thymus-derived cell in the 6. Noma,Y., Sideras, T., Naito, T., Berginitiation of the immuneresponse? stedt-Lindqvist, A., Azuma,C., SeverProg. Immunol.1:355-68 inson, E., Tanabe,T., Kinashi,T., Mat2. Schimpl,A., Wecker,E. 1972.Replacesuda, F., Yaoita, Y., Honjo,T. 1986. mentof T-cell function by a T-cell Cloning of eDNA encoding the murine product. Nature 237:15-17 IgG1inductionfactor by a novel strat3. Kishimoto,T., Miyake,T., Nishizawa, egy using SP6 promoter. Nature 319: 640-46 Y., Watanabe,T., Yamamura, Y. 1975. Triggering mechanismof B lympho7. Lee, F., Yokota,T., Otsuka,T., Meycytes. I. Effect of anti-immunoglobulin erson, P., Villaret, D., Coffman,R., and enhancing soluble factor on Mosmann,T., Rennick, D.,. Roeham, differentiation andproliferation of B N., Smith, C., Zlotnick, A., Arai, K. cells. J. Immunol.15:117%84 1986.Isolation andcharacterization of 4. Parker,D. C., Fothergill, J. J., Wadsa mouseinterleukin cDNA clone that worth, D. C. 1979. B lymphocyteactiexpresses B-cell stimulatory factor 1 vation by insoluble anti-immunoactivities andT-cell and mast-cell-stiglobulin: Induction of immunomulatingactivities. Proc. Natl. Acad. globulin secretion by a T cell-depenSci. USA83:2061-65

Annual Reviews

Annu. Rev. Immunol. 1988.6:485-512. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

BSFs ANDRECEPTORS 8. Kinashi,T., Harada,N., Severison,E., Tanabe,R., Sideras, P., Konishi, M., Azuma,C., Tominaga,A., BergstedtLindqvist, S., Takahashi, M., Matsuda,F., Yaoita, Y., Takatsu, K., Honjo, T. 1986. Cloning of complementary DNAencoding T-cell replacing factor and identity with Bcell growthfactor II. Nature324: 7073 9. Hirano, T., Yasukawa,K., Harada,H., Taga, T., Watanabe,Y., Matsuda,T., Kashiwamura,S., Nakajima,K., Koyama, K., Iwamatsu,A., Tsunasawa,S., Sakiyama,F., Matsui, H., Takahara, Y., Taniguchi,T., Kishimoto,T. 1986. Complementary DNAfor a novel human interleukin (BSF-2) that induces B lymphocytes to produce immunoglobulin.Nature 324:73-76 10. Paul, W.E., Ohara,J. 1987.B cell stimulatoryfactor 1/Interleukin 4. Ann. Re~. lmmunoL5:429-59 11. Muraguchi,A., Kishimoto, T., Miki, Y., Kuritani,T., Kaieda,T., Yoshizaki, K., Yamamura, Y. 1981. T cell replacing factor (TRF)-induced IgGsecretion in a humanB blastoid cell line and demonstrationofacceptorsfor TRF.J. Imrnunol. 127:412-16 12. Kishimoto,T., Yoshizaki,K., Kimoto, M., Okada,M., Kuritani, T., Kikutani, H., Shimizu, K., Nakagawa,T., Nakagawa, N., Miki, Y., Kishi, H., Fukunaga, K., Yoshikubo, T., Taga, T. 1984.Bcell growthand differentiation factors and mechanism of B cell activation. Immunol.Rev. 78:97-118 13. Okada, M., Sakaguchi, N., Yoshimura,N., Hara, H., Shimizu,K., Yoshida, N., Yoshizaki, K., Kishimoto, S., Yamamura,Y., Kishimoto, T. 1983. B cell growth factor (BCGF) and B cell differentiation factor from human T hybridomas. Twodistinct kinds of BCGFs and their synergismin B cell proliferation. J. Exp.Med.157: 583-90 14. Butler, J. L., Falkoff, R. J. M., Fauci, A. S. 1984. Developmentof a human T-cell hybridomasecreting separate Bcell growthanddifferentiation factors. Proc. NatLAcad. Sci. USA81: 247578 15. Yoshizaki, K., Nakagawa,T., Kaieda, T., Muraguchi, A., Yamamura,Y., Kishimoto,T. 1982. Induction of proliferation and Igs-productionin human B leukemic ceils by anti-immunoglobulins andT cell factors. J. Irnrnunol. 128:1296-1301 16. Kikutani, H., Taga, T., Akira, S., Kishi, H., Miki, Y., Saiki, 0., Yama-

507

mura,Y., Kishimoto,T. 1985.Effect of B cell differentiation factor (BCDF) biosynthesis and secretion of immunoglobulin moleculesin humanB cell lines. J. Immunol.134:990-95 17. Saiki, O., Ralph, P. 1983. Clonal differencesin responseto T cell replacing factor for IgMsecretion and TRF receptors in a humanB lymphoblast cell line. Eur.J. lrnmunol.13:31-34 18. Shimizu,K., Hirano,T., Ishibashi, K., Nakano,N., Taga, T., Sugamura,K., Yamamura,Y., Kishimoto, T. 1985. Immortalization of BGDF(BCGFII)and BCDF-producing T cells by humanT cell leukemia virus (HTLV) and characterization of humanBGDF (BCGFII). J. lmmunol. 134: 172833 19. Hirano, T., Taga, T., Nakano, N:, Yasukawa, K., Kashiwamura, S., Shimizu, K., Nakajima,K., Pyun, K. H., Kishimoto,T. 1985.Purification to homogeneityand characterization of humanB cell differentiation factor (BCDF of BSFp-2). Proc. Natl. Acad. Sci. USA82:5490-94 20. Hirano, T., Taga, T., Yasukawa,K., Nakajima,K., Nakano,N., Takatsuki, F., Shimizu, M., Murashima,A., Tsunasawa,S., Sakiyama,F., Kishimoto, T. 1987. Human B cell differentiation factor definedby an anti-peptide antibodyand its possible role in autoantibodyproduction.Proc. Natl..4cad. Sci. USA84:228-31 21. Paul, W.E. 1983. Proposednomenclature for B-cell stimulating factors. Immunol. Today 4:332-32 22. Yasukawa,K., Hirano, T., Watanabe, Y., Muratani,K., Matsuda,T., Nakai, S., Kishimoto,T. 1987. Structure and expressionof humanB cell stimulatory factor-2 (BSF2/IL-6)gene. EMBO. J. 6:2939-45 23. Nagata, S., Tsuchiya, M., Asano,S., Yamamoto,O., Hirata, Y., Kubota, N., Oheda, M., Nomura, H., Yamazaki, T. 1986. The chromosomalgene structure and two mRNAs for human granulocytecolony-stimulatingfactor. EMBO.J. 5:575-81 24. Sato, T., Matsui, H., Shibahara, S., Kobayashi, T., Morinaga, Y., Kashima, N., Yamasaki, S., Hamuro, J., Taniguchi,T. 1987. Newapproaches for the high-level expressionof human interleukin-2 cDNAin E. coli. J. Biochern. 101:525-34 25. Taga, T., Kawanishi,Y., Hardy,T. R., Hirano, T., Kishimoto,T. 1987. Receptors for B cell stimulatory factor 2 (BSF2):quantitation,.specificity, dis-

Annual Reviews

Annu. Rev. Immunol. 1988.6:485-512. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

508

KISHIMOTO& HIRANO

tribution and regulation of the expression.J. Exp. Med.In press 25a. Muraguchi,A., Hirano, T., Tang,B., Matsuda,T., Horii, Y., Nakajima,K., Kishimoto,T. 1988.Theessential role of B cell stimulatory factor 2 (BSF2/IL-6) for the terminaldifferentiation of B cells. J. Exp. Med.In press 26. VanSnick, J., Vink, A., Cayphas,S., Uyttenhove,C, 1987. Interleukin-HPl, a T cell-derived hybridoma growthfactor that supportsthe in vitro growthof murine plasmacytomas.J. Exp. Med. 165:641~,9 27. Van Damme,J., Opdenakker, G., Simpson, R. J., Rubira, M. R., Cayphas,S., Vink, A., Billiau, A., Van Snick, J. 1987. Identification of the human26-kDprotein, interferon f12 (IFN-fi2), as a B cell hybridoma/ plasmacytomagrowth factor induced by interleukin 1 and tumor necrosis factor. J. Exp. Med.165:914-19 28. Van Damme, J., Cayphas, S., Opdenakker, G., Billiau, A., Van Snick, J. 1987. Interleukin 1 and poly(rI) poly(rC)induceproduction a hybridomagrowth factor by human fibroblasts. Eur.J. lrnmunol.17:1-7 29. Nordan, P. R., Potter, M. 1986. A macrophage-derived factor required by plasmacytomasfor survival and proliferation in vitro. Science233:566-69 30. Namba,Y., Hanaoka, M. 1972. Immunologyof cultured IgM-formingcells of mouse.I. Requirement of phagocytic cell factor for the growthof IgM-forming tumorceils in tissue culture. J. Immunol. 109:1193-1200 31. Corbel,C., Melchers,F. 1984.Thesynergismof accessorycells andof soluble alpha-factors derived fromthemin the activation of Bceils to proliferation. lmmunol.Rev. 78:51-74 32. Shrestha, K. R., Hiramoto, N., Ghanta, V. K. 1984. Regulation of MOPC 104Eby T cells and growthfactors inducedby C. parvumstimulation. lnt. J. Cancer33:845-50 33. Astaldi, G. C. B., Janssen, M. C., Lansdorp, P., Willems, C., Zeijlemaker, W. P., Oosterhof, F. 1980. Humanendothelial culture supernatant (HECS):a growth factor for hybridomas.J. Immunol.125:1411-14 34. Aarden,L., Lansdorp,P., Degroot,E. 1985. A growthfactor for B cell hybridomas produced by human monocytes. Lymphokines10:175-85 35. VanSnick, J., Cayphas,S., Vink, A., Uyttenhove,C., Coulie, P. G., Rubira, M. R., Simpson, R. J. 1986. Purification and NH2-terminalaminoacid

sequence of a T-cell-derived lymphokinewith growthfactor activity for B-cell hybridomas.Proc. Natl. Acad. Sci. USA83:9679-83 36. Potter, M., Boyce,C. 1962. Induction of plasma cell neoplasms in strain Balb/c mice with mineral oil and mineral oil adjuvants. Nature 193: 1086-87 37. Weissenbach,J., Chernajovsky, Y., Zeevi, M., Shulman,L., Sorequ, H., Nir, U., Wallach,D., Perricaudet, M., Tiollais, P., Revel,M.1980.Twointerferon mRNAs in humanfibroblasts. In vitro translation and Escherichiacoli cloningstudies. Proc.Natl. Acad.Sci. USA77:7152-56 38. Content, J., DeWit, L., Poupart, P., Opdenakker, G., Van Damme,J., Billiau, A. 1985. Induction of a 26kDa-proteinmRNA in humancells treated with an interleukin-l-related, leukocyte-derived factor. Eur. J. Biochem.152:253-57 39. Sehgal,P. B., Sagar, A. D. 1980.Heterogeneityof poly(I) ¯ poly(C)-induced human fibroblast interferon mRNA species. Nature288:95-97 40. Kohase,M., Henriksen-DeStefano,D., May,L. T., Vilcek, J., Sehgal, P. B. 1986. Induction of fi2-interferon by tumor necrosis factor. A homeostatic mechanism in the control of cell proliferation. Cell 45:659~6 41. Haegeman, G., Content, J., Volckaert, G., Dery~ack,R., Tavernier,J., Fiers, W. 1986. Structural analysis of the sequencecoding for an inducible 26kDa protein in humanfibroblasts. Eur. J. Biochem. 159:625-32 42. Zilberstein, A., Ruggieri,R., Korn,J. H, Revel, M. 1986. Structure and expression of cDNAand genes for humaninterferon-/~2, a distinct species inducible by growth-stimulatorycytokines. EMBO J. 5:2529-37 42a. Hirano, T., Matsuda,M., Hosoi, K., Okano,A., Matsui, H., Kishimoto,T. 1987. Absenceof antiviral activity in recombinantB cell stimulatory factor 2 (BSF-2).ImmunoL Lett. In press 43. Poupart, P., Vandenabeele, P., Cayphas,S., VanSnick, J., Haegeman, G., Kruys,V., Fiers, W., Content, J. 1987. B cell growth modulating and differentiating activity of recombinant human26-Kdprotein (BSF-2, HulFN/~2, HPGF).EMBO J. 6:1219-24 43a. Ikebuchi, K., Wong,G. G., Clark, S. C., Ihle, J. N., Hirai, Y., Ogawa,M. 1987. Interleukin-6 enhancementof interleukin-3-dependentproliferation of multipotential hemopoietic pro-

Annual Reviews

Annu. Rev. Immunol. 1988.6:485-512. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

BSFs ANDRECEPTORS genitors. Proc. Natl. Acad.Sci. USA. In press 44. Gauldie,J., Richards,C., Harnish,D., Lansdorp, P., Baumann, H. 1987. Interferon fl2/BSF-2 shares identity with monocytederived hepatocytestimulating factor (HSF)and regulates the majoracute phase protein response in liver cells. Proc. Natl. Acad.Sci. USA.In press 45. Andus, T., Geiger, T., Hirano, T., Northoff, H., Ganter, U., Bauer, J., Kishimoto,T., Heinrich, P. C. 1987. RecombinanthumanB cell stimulatory factor 2 (BSF2/IFN-fl2)regulates /~fibrinogen and albumin mRNA levels in Fao-9cells. FEBSLett. In press 46. Kushner,I., Volonakis,J. E., Gewurz, H. 1982. C-reactive protein and the plasma protein response to tissue injury. Ann. N.Y. Acad.Sci. 389: 3948 47. Ramadori,G., Sipe, J. D., Dinarello, C. A., Mizel,S. B., Colten,H.R. 1985. Pretranslational modulationof acute phase hepatic protein synthesis by murinerecombinantinterleukin 1 (ILl) and purified humanIL-1. J. Exp. Med. 162:930-42 48. Ritchie, D. G., Fuller, G. M. 1983. Hepatocyte-stimulating factor: a monocyte-derived,acute-phase regulatory protein. Ann. N.Y. Acad.Sci. 408:490-502 49. Baumann, H., Hill, R. E., Sauder, D. N., Jahreis, G. P. 1986. Regulationof major acute-phase plasmaproteins by hepatocyte-stimulating factors of humansquamouscarcinoma cells. J. Cell. Biol. 102:370-83 50. Greene, L. A., Rein, G. 1977. Synthesis,storage, andrelease of acetylcholine by a nerve-growthfactor responsive line of rat phenochromocytoma cells. Nature268: 34951 51. Gurney,M.E., Heinrich,S. P., Lee,M. R., Yin, H. 1986. Molecular cloning and expression of neuroleukin, a neurotropicfactor for spinal and sensory neurons. Science 234:566-74 52. Gurney,M. E., Apatoff, B. R., Spear, G. T., Baumel,M. J., Antel, J. P., Bania, M. B., Reder, A. T. 1986. Neuroleukin:A lymphokineproduct of lectin-stimulatedT cells. Science234: 574-81 53. Howard,M., Farrar, J., Hilfiker, M., Johnson, B., Takatsu, K., Hamaoka, T., Paul, W.E. 1982.Identification of a T cell-derived B cell growthfactor distinct from interleukin 2. J. Exp. Med. 155:914-23

509

54. Ohara, J., Paul, W.E. B cell stimulatory factor BSF1. 1985. Production of a monoclonalantibody and molecular characterization. Nature 315:333-36 55. Ohara, J., Lahet, S., Inman,J., Paul, W.E. 1985. Partial purification of BSF I by HPLC.J. Immunol.135:2518-23 56. Rabin, E. M., Ohara, J., Paul, W.E. 1985.B cell stimulatoryfactor (BSF)1 activates resting B cells. Proc.Natl. Acad. Sci. USA82:2935-39 57. Noelle, R., Krammer, P. H., Ohara,J., Uhr, J. W., Vitetta, E. S. 1984. Increasedexpressionof Ia antigens on resting Bcells. Anadditional role for B-cell growthfactor. Pro¢.Natl. Acad. Sci. USA81:6149-53 58. Rabin, E. M., Mond,J. J., Ohara,J., Paul, W.E. 1986. B cell stimulatory factor (BSF)-Iprepares resting Bcells to enter S phase in responseto antiIgMand to lipopolysaccharide.J. Exp. Med. 164:517-31 59. Vitetta, E. S., Brooks, K., Chen, Y. W., Isakson,P., Jones, S., Layton,J., Mishra, G. C., Pure, E., Weiss, E., Word,C., Yuan,D., Tucker, P., Uhr, J. W., Krammer,P. H. 1984. T cellderived lymphokinesthat induce IgM and IgG secretion in activated murine B ceils. Immunol.Rev. 78:137-57 60. Siderase, P., Bergstedt-Lindqvist,S., Severinson, E. 1985. Partial biochemical characterization of IgG1inducing factor. Eur. J. Immunol.15: 593-98 61. Vitetta, E. S., Ohara, J., Myers,C., Layton, J., Krammer,P. H., Paul, W. E. 1985. Serological, biochemical,and functional identity of B cell-stimulatoryfactor 1 and B cell differentiation factor for IgG1.J. Exp. Med. 162:1726-31 62. Hamaguchi,Y., Kanakura,Y., Fujita, J., Takeda,S., Nakano,T., Tarui, S., Honjo,T., Kitamura,Y. 1987. Interleukin 4 as an essential factor for in vitro clonal growth of murine connective tissue-type mastcells. J. Exp. Med. 165:268-73 63. Yokota,T., Otsuka, T., Mosmann, T., Banchereau, J., DeFrance,T., Blanchard,D., Vries,J. E. D., Lee,F., Arai, K. 1986.Isolation andcharacterization of a humaninterleukin cDNAdone, homologousto mouseB-cell- and Tcell-stimulatingactivities. Proc.Natl. Acad. Sci. USA83:5894-98 64. Kikutani, H., Inui, S., Sato, R., Barsumian, E. L., Owaki,H., Yamasaki, K., Kaisho, T., Uchibayashi, N., Hardy, R. R., Hirano, T., Tsunasawa,

Annual Reviews

Annu. Rev. Immunol. 1988.6:485-512. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

510

KISHIMOTO& HIRANO

S., Sakiyama,F., Suemura,M., Kishimoto, T. 1986. Molecularstructure of human lymphocyte receptor for immunoglobulinE. Cell 47:657~5 65. Yoshizaki, K., Nakagawa,T., Fukunaga, K., Kaieda, T., Maruyama,S., Kishimoto, S., Yamamura, Y., Kishimoto, T. 1983. Characterization of humanB cell growth factor (BCGF) from a clonedT cell or mitogen-stimulated T cells. J. ImmunoL 130: 124146 66. Swain,S. K., Dutton,R. W.1982. Production of a B cell growth-promoting activity, (DL) BCGF,from a cloned cell line and its assay on the BCL1B cell tumor. J. Exp. Med. 156: 182134 67. Swain,S. L., Howard,M., Kappler,J., Marrack, P., Watson,J., Booth, R., Wetzel, G. D., Dutton, R. W. 1983. Evidencefor two distinct classes of murine B cell growth factors with activities in differentfunctionalassays. J. Exp. Med.158:822-35 68. Pike, B. L., Vaux,D. L., Clark-Lewis, I., Schrader, J. W., Nossal, G. J. V. 1982.Proliferation and differentiation of single laapten-specificBlymphocytes is promotedby T-cell factor(s) distinct fromT-cell growthfactor. Proc. Natl. Acad. Sci. USA79:6350-54 69. Harada, N., Kikuchi, Y., Tominaga, A., Takaki, S., Takatsu, K. 1985. BCGF II activity on activated B cells of a purified murineT cell-replacing factor (TRF)from a T cell hybridoma (B151K12). J. ImmunoL134: 394451 70. Takatsu, K., Kikuchi, Y., Takahashi, T., Honjo, T., Matsumoto, M., Harada, N., Yamaguchi, N., Tominaga, A. 1987.Interleukin 5, a T-cellderived B-cell differentiation factor also induces cytotoxic T lymphocytes. Proc. NatL Acad. Sci. USA84: 423438 71. Sanderson,C. J., O’Garra,A., Warren, D. J., Klaus, G. G: B. 1986. Eosinophil differentiation factor also has B-cell growthfactor activity: Proposedname interleukin 4. Proc. Natl. Acad. Sci. USA83:437-40 72. Harada, N., Takahashi, T., Matsumoto,M., Kinashi, T., Ohara,J., Kikuchi, Y., Koyama,N., Severinson, E., Yaoita, Y., Honjo,T., Yamaguchi, N., Tominaga,A., Takatsu, K. 1987. Production of a monoclonalantibody to and its use in the molecular characterization of murineT cell-replacing factor (TRF)and B cell growthfactor II (BCGFII). Proc. NatL Acad. Sci.

USA 84:4581-85 73. Mosmann, T. R., Coffman,R. L. 1987. Twotypes of mousehelper T-cell clone. Immunol. Today 8:223-27 74. Azuma,C., Tanabe,T., Konishi, M., Kinashi, T., Noma,T., Matsuda,F., Yaoita, Y., Takatsu, K., Hammarstrrm, L., Smith,C. I. E., Severinson, E., Honjo, T. 1986. Cloning of cDNA for humanT-cell replacing factor (intedeukin-5) and comparisonwith the murine homologue.Nucleic Acids. Res. 14:9149-58 74a. Sharma,S., Mehta, S., Morgan,J., Maizel,A. 1987.Molecularcloning and expression of a humanB-cell growth factor genein Escherichiacoli. Science 235:1489-92 75. Leonard, W.J., Depper, J. M., Crabtree, G. R., Rudikoff,S., Pumphrey, J., Rabb,R. J., Krrnke,M., Svetlik, P. B., Peffer, N. J., Waldmann, T. A., Greene, W.C. 1984b. Molecular cloning and expression of cDNAsfor the human interleukin-2 receptor. Nature 311: 626-31 76. Nikaido, T., Shimizu,A., Ishida, N., Sabe, H., Teshigawara,K., Maeda,M., Uchiyama,T., Yodoi, J., Honjo, T. 1984. Molecular cloning of cDNA encodinghumaninterleukin 2 receptor. Nature 311:631-35 77. Ohara,J., Paul, W.E. 1987. Receptors for B-cell stimulatory factor 1 expressedon cells of haematopoieticlineage. Nature 325:53740 78. Noma,T., Mizuta, T., Rosen, A., Hirano, T., Kishimoto,T., Honjo, T. 1987. Enhancement of the interleukin 2 receptor expressionon T cells by multiple B lymphotropic lymphokines. ImmunoLLett. 15:249-53 79. Tsudo, M., Kozak, R. W., Goldman, C. K., Waldmann,T. A. 1986. Demonstration ofa non-Tacpeptide that binds interleukin 2: A potential participant in a multichaininterleukin 2 receptor complex.Proc. Natl. Acad. Sci. USA 83:9694-98 80. Teshigawara,K., Wang,H., Kato, K., Smith,K. A. 1987. Interleukin 2 highaffinity receptor expression requires two distinct bindingproteins. J. Exp. Med. 165:223-38 81. Johnson, D., Lanahan, A., Buck, C. R., Sehgal,A., Morgan,C., Mercer,E., Bothwell, M., Chao, M. 1986. Expressionand structure of the human NGFreceptor. Cell 47:545-54 82. Park, L. S., Friend, D., Grabstein,K., Urdal, D. L. 1987. Characterizationof the high-affinity cell surface receptor for murineB-cell-stimulatingfactor 1.

Annual Reviews

Annu. Rev. Immunol. 1988.6:485-512. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

BSFs ANDRECEPTORS 51 1 Proc. Natl. Acad.’Sci. USA84: 166973 83. Nakajima, K., Hirano, T., Koyama, K., Kishimoto,T. 1987. Detection of receptors for murineBcell stimulatory factor 1 (BSFI):Presenceof functional receptors on CBA/N splenic B cells. J. Immunol. 139:774 84. Howard,M., Matis, L. A., .Malek, T. R., Shevach,E. M., Kell, W., Cohen, D., Nakaoishi, K., Paul, W.E. 1983. Interleukin 2 inducesantigen-reactive T cell lines to secrete BCGF-I. J. Exp. Med. 158:2024-39 85. Yakura,H., Kawabata,I., Ashida,T., Shen, F. W., Katagiri, M.1986. A role of Lyb-2in B cell activation mediated bya Bcell stimulatoryfactor. J. Irnmunol. 137:1475-81 86. Mishra,G. C., Berton, M. T., Oliver, K. G., Krammer,P. H., Uhr, J. W., Vitetta, E. S. 1986.A monoclonal antimouse LFA-I~ antibody mimics the biologicaleffects of B cell stimulatory factor-1 (BSF1). J. Immunol. 137: 1590-98 87. Chen, Z. Z., Coggeshall,K. M., Cambier, J. C. 1986. Translocationof protein kinase C during membrane immunoglobulin-mediated transmembrane signaling in B lymphocytes.J. lmmunoL 136:2300-2304 88. Weiss, A., Imboden,J., Hardy, K., Hanger, B., Terhorst, C., Stobo, J. 1986.Therole of the T3/antigenreceptor complexin T cell activation. Ann. Rev. lmrnunol. 4:593~19 89. Maino, V. C., Hayman, M. J., Crumpton,M. J. 1975. Relationship betweenenhanced turnover of phosphatidylinositol and lymphocyteactivation by mitogen. Biochem.J. 146: 247-52 90. Ransom,J. T., DiGiusto,D. L., Cambier, J. C. L. 1985.Singlecell analysisof calciummobilization in anti-receptor antibodystimulated B lymphocytes.J. ImmunoL136:54-57 91. Mizuguchi,J., Beaven,M.A., Ohara, J., Paul, W.E. 1986. BSF1action on resting B cells does not require elevation of inositol phospholipidmetabolismor increased(CaZ+).J. Immunol. 137:2215-19 92. Cambier, J. C., Ransom,J. T. 1987. Early events in B lymphocyteactivation. Ann. Rev. ImmunoL5:175-99 93. Mond,J. J., Carman,J., Sarma, C., Ohara, J., Finkelman, F. D. 1986. Interferon-), suppressesB cell stimulation factor (BSF-1) inductionof class II MHC determinants on B cells. J. lmmunoL137:3534-37

94. Ullrich, A., Bell, J. R., Chen,E. Y., Herrera,R., Petruzzelli,L. M., Dull, T. J., Gray,A., Coussens,L., Liao, Y.-C., Tsubokawa,M., Mason,A., Seeburg, P. H., Grunfeld, C., Rosen, O. M., Ramachandran,J. 1985. Humaninsulin receptorandits relationship to the tyrosine kinase family of oncogenes. Nature 313: 7564il 95. Sutton, M.G. S. J., Mercier,L., Giuliani, E. R., Lie, J. T. 1980.Atrial Myxomas.Areviewof clinical experiencein 40 patients. MayoClin. Proc. 55: 37176 96. AI-Balaghi,S., Str6m,H., M611er,E. 1984.B cell differentiation factor in synovial fluid of patients with rheumatoidarthritis. Immunol.Rev. 78: 723 97. Castleman,B., Iverson, L., Menendez, V. P., Menendez,V. P. 1956. Localized mediastinal lymphonodehyperplasia resemblingthymoma.Cancer9: 822 98. Coffman,R. L., Ohara, J., Bond, M. W.,Carry,J., Zlotnick,E., Paul, W.E. 1981. B cell stimulatory factor 1 enhances the IgE response of lipopolysaccharide-activated B cells. J. ImrnunoL136:4538-41 99. Finkelman,F. D., Katona, I., Urban, J. F., Snapper,C. M., Ohara,J., Paul, W. E. 1986. Suppression of in vivo polyclonal IgE responses by monodonal antibody to the lymphokine BSF1.Proc. NatLAcad. Sci. USA83: 9675-78 100. Mosmann,T. R., Cherwinski, H., Bond,M. W., Giedlin, M.A., Coffman, R. L. 1986.Twotypes of murinehelper T cell clone. I. Definitionaccordingto profiles of lymphokineactivities and secreted proteins. J. Immunol.136: 2348-57 101. Kishimoto, T., Ishizaka, K. 1973. Regulations of antibody response in vitro. VI. Carrier-specifichelper cells for IgGand IgE antibody response. J. ImmunoL111:720-32 102. Kishimoto, T., Ishizaka, K. 1973. Regulation of antibody response in vitro. VII. Enhancingsoluble factors for IgGand IgE antibodyresponse. J. Immunol. 111:1194-1205 103. Snapper, C. M., Paul, W. E. 1987. Interferon-), andBcell stimulatoryfactor-I reciprocally regulate Ig isotype production. Science 236:944-47 104. Suemura,M., Ishizaka, A., Kobatake, S., Sugimura, K., Maeda,K., Nakanishi, K., Kishimoto,S., Yamamura, Y., Kishimoto,T. 1983. Inhibition of IgE production in B hybridomas by IgE

Annual Reviews 512

KISHIMOTO& HIRANO

Annu. Rev. Immunol. 1988.6:485-512. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

class-specific suppressorfactor fromT hybridomas.J. Irnmunol. 130: 105660 105. Hirashima,M., Yodoi,J., Ishizaka, K. 1980. Regulatoryrole of IgE-binding factors from rat T lymphocytes.III. IgE-specific suppressive factors with IgE-bindingactivity. J. lmmunol.125:

1442-48 106. Perussia, B., Dayton,E. T., Lazarus, R., Fanning,V., Trinchieri, G. 1983. Immune interferon induces the receptor for monomeric IgG1 on human monocyticand myeloid cells. J. Exp. Med. 158:1092

Annu. Rev. Immunol. 1988.6:485-512. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Annu. Rev. Immunol. 1988.6:485-512. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Annual Reviews www.annualreviews.org/aronline

Annu. Rev. Immunol. 1988.6:513-534. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Ann.Rev. lmmunol.1988.6 .’513-34 Copyright©1988by AnnualReviewsInc. All rights reserved

IgE-BINDING FACTORS AND REGULATION OF THE IgE ANTIBODY RESPONSE Kim&hige Ishizaka Subdepartmentof Immunology,Johns Hopkins University School of Medicine, Baltimore, Maryland21205 INTRODUCTION IgE-bindingfactors were first detected in culture supernatants of mesenteric lymphnode (MLN)cells of rats infected with Nippostrongylus brasiliensis (Nb), by the ability of the factors to inhibit rosette formation of Fc, R+ lymphocyteswith IgE-coatederythrocytes (1). The factors had an affinity for homologous IgE and could be purified by using IgE-coupled Sepharose(2). Culture supernatants of normalMLN cells did not contain a detectable amountof IgE-bindingfactor (IgE-bF); however,incubation" of the cells with homologous IgE resulted in the formationof IgE-bF.The majorcell sourceof IgE-bFin the systemwasT cells. Normalrat T cells, whichcontained less than 0.1%B cells, formedIgE-bF uponincubation with IgE (1). Subsequentexperimentsrevealed that the T ce!l-derived IgE-bFconsisted of two types; one type selectively enhanced the in vitro IgE response without affecting the IgG response, while another type selectively suppressed the IgE response (2, 3). These findings suggested that IgE-bF wouldbe effector lymphokinesthat selectively regulate the IgE response in an isotype-specific manner.Thus, ! included basic findings on IgE-bF in mypreviousreviewon the regulation of IgEsynthesis(4). In this current review, I briefly summarizebiologic activities of the factors and then describe further progress on molecularcharacterization of the T cellderived IgE-bFand Fc, R on B cells, of whichfragmentsmayhave affinity for IgE. Cellular and molecularmechanisms for the selective formationof IgE-potentiating factor (IgE-PF) or IgE-suppressivefactor (IgE-SF) 513 0732-0582/88/0410-0513502.00

Annual Reviews 514 ISHIZAKA lymphokines involved in the mechanismsare discussed. Evidence is presented that a subset of antigen-primed T cells form IgE-bF upon antigenic stimulation and that these "regulatory" T cells mayplay important roles in the regulation of the IgE response.

Annu. Rev. Immunol. 1988.6:513-534. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

CORRELATION BETWEEN THE IgE ,RESPONSE AND BIOLOGIC ACTIVITIES OF T CELL-DERIVED IgE-bF A series of experiments indicated that selective enhancementof the IgE response by various immunological maneuvers was accompanied by the formation of IgE-PF, while procedures for suppressing the IgE response induced the formation of IgE-SF. Thus, infection of rats with Nb, which selectively enhances the IgE synthesis, induces the formation of IgE-PF (3). A single injection of Bordetella pertussis vaccine, which is the best adjuvant for the IgE response, also induces the formation of IgE-PF (5). In contrast, repeated injections of complete Freund’s adjuvant (CFA), which is knownto selectively suppress the IgE response to an unrelated antigen (6), induce the formation of IgE-SF (7). It was also found formation of IgE-bF is associated with the immuneresponse. Immunization of Lewis strain rats with keyhole limpet hemocyanin (KLH) absorbed to aluminum-hydroxide gel (alum) resulted in the formation of IgE antibodies, and their spleen cells formed IgE-PF upon antigenic stimulation. However, when the same strain was immunized with KLH included in CFA, they did not develop any IgE antibody response, and their spleen cells formed IgE-SF upon antigenic stimulation (8). Similar findings were observed in the mouse as well. Whenhigh IgE producer strain, BDF1 mice were immunized with a minimum dose of alumabsorbed ovalbumin (OA) for a persistent IgE antibody formation, their spleen cells formed IgE-PF upon antigenic stimulation (9). In contrast, intravenous injections of ovalbumin into the same strain suppressed the IgE antibody response to alum-absorbed OA(10) and primed their spleen cells for the formation of IgE-SF (11). Kishimotoet al (12) described priming of BALB/cmice with DNP-derivatives of mycobacteria (DNPMyc) which suppressed the IgE antibody response to alum-absorbed DNPOA, and indicated that spleen cells of DNP-Myc-primedmice released IgE-specific suppressive factor (IgE-TsF) upon incubation with DNPbovine serum albumin (BSA) (13). Their subsequent experiments showed that IgE-TsF had affinity for IgE and belonged to IgE-bF (14). The magnitude of the IgE response and the biologic activities of IgEbF differ, depending on mouse strains. In contrast to BDF1mice, SJL mice when immunized with alum-absorbed antigen were induced to form

Annual Reviews

Annu. Rev. Immunol. 1988.6:513-534. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

IgE-BINDING FACTORS

515

IgGantibodies but essentially no IgE antibodies, and incubation of their spleen cells with the antigen resulted in the formationof IgE-SF(9). Such strain differences were observedevenwithout immunization.Whensplenic lymphocytes(or MLN cells) of Lewis strain rats or BALB/cmice were incubatedwith homologous IgE, IgE-bFformedby the cells wasa mixture of IgE-PFand IgE-SF.Incubation of spleen cells of a high IgE producer, BDF1mice, with mouseIgE resulted in the formation of IgE-PF. In contrast, normal SJL mousespleen cells formedIgE-SFupon incubation with IgE (9, 15). It is knownthat low doseX-ray irradiation or treatment with cyclophosphamide selectively enhancesthe IgE response (16). Even SJLmice producedIgE antibodies if they received low dose X-irradiation or cyclophosphamideprior to immunization,and spleen cells of these treated animals formed IgE-PF upon incubation with the homologous antigen (17). Thesefindings collectively indicate that IgE-PFis formed wheneverIgE synthesis is enhanced and/or IgE antibody response is induced, while IgE-SFis formedin various conditions in whichthe IgE responseis suppressed. Thesameprinciples maybe applied to humanIgE-bF.Saryan et al (18) found that T cells of patients with hyper-IgEsyndrome,such as atopic dermatitis, constitutively secrete IgE-bFwhichselectively enhancespontaneous formation of IgE by lymphocytesof atopic individuals. Leunget al (19) reported that serumof nonatopicindividuals, whohave extremely low serumIgE levels, containedIgE-bFwhichselectively suppressedthe IgE-formationby peripheral blood lymphocytesof atopic patients. The correlation betweenan enhancement of the IgE response and the formation of IgE-PF, and that between suppression of the IgE response and the formation of IgE-SF, strongly suggest that IgE-bF are involved in the regulation of the IgE responsein vivo. PHYSICOCHEMICAL PROPERTIES STRUCTURE OF IgE-bF

AND

TheT cell~terived IgE-bFare glycopeptides and are heterogeneouswith respect to their molecularweight.When spleen cells of antigen-primedrats or mice were stimulated by homologousIgE, the IgE-bF formedby the cells consisted of the 60-kd, 30-kd and 15-kdspecies (15). RodentT cell hybridomas,i.e. 23B6and 231F1(20, 21) as well as humanT cell hybridoma166A2(22), formedthe three species (by weight) whenthey incubated with homologousIgE. Another rodent hybridoma, 23A4(23), and humanT cell hybridoma, 166G11and 400G2(22), formed the 60-kd and 30-kd IgE-bFuponincubation with IgE. Younget al (24) established

Annual Reviews

Annu. Rev. Immunol. 1988.6:513-534. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

516 ISHIZAKA a humanT cell clone from peripheral blood of an atopic dermatitis patient. The T cell clone constitutively secreted the 60-kd and 15-kd IgE-PF (25). The major differences between the 15-kd IgE-PF and the 15-kd IgE-SF appear to be carbohydrate moieties. The 15-kd IgE-PF from both rodent T cells and humanT cell hybridomashave affinity for lentil lectin and Con A (4, 22). The 15-kd IgE-SFfrom both species failed to bind to the lectins but had affinity for peanut agglutinin (PNA)(22, 26). Although the 60-kd IgE-SF do not necessarily bind to PNA(20), the 60-kd IgE-PF from both species haveaffinity for lentil lectin. The amino acid sequence of the rodent IgE-bF peptide was revealed by gene cloning. Martens et al (27) incubated the rodent T cell hybridoma 23B6 with rat IgE to induce the formation of IgE-SF (23), and they obtained mRNAfrom the cells. Since the injection of the mRNA into Xenopaslaevis oocyte resulted in the formation of IgE-bF, they constructed eDNAlibraries from the mRNA and isolated four cDNAclones encoding IgE-bF. Transfection of COS7 monkeykidney cells with the cDNAclone in mammalian cell expression vector pcD resulted in the formation of IgEbF. None of the IgE-bF derived from the four eDNAclones suppressed the in vitro IgE response, but the prodtiets of two eDNA clones selectively potentiated the IgE response. The IgE-bF derived from one of the eDNA clones, i.e. clone 8.3, consisted of two molecular weight species of about 60 kd and 11 kd. Both species of the IgE-bF had affinity for lentil lectin and selectively potentiated the IgE response. The nucleotide sequence of eDNAclone 8.3 revealed a putative protein coding region of 556 amino acids (Figure 1). The peptide contained two potential sites for N-linked glycosylation (CHOsite) and several potential sites for posttranslational proteolytic cleavage (shownby arrows in Figure 1). The molecular weight of the peptide calculated from the predicted amino acid sequence was approximately 62,000 and corresponded to the 60-kd IgE-bF formed by transfection with the cDNAclone. Thus, the 11-

pcD8.3

Figure 1 Restriction map of pcD clone 8.3 encoding rodent IgE-binding factor. Bottom bar indicates the putative protein coding region. Potential N-linked glycosylation site (CHO) and proteolytic cleavage sites (arrows) are also shown.

Annual Reviews

Annu. Rev. Immunol. 1988.6:513-534. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

IgE-BINDING

FACTORS 517

kd IgE-PF must be a cleavage product of the 60-kd peptide. The 11-kd IgE-PFhas affinity for lentil lectin, indicating that the molecules contain one of the two sites for N-glycosylation. Martens et al (28) constructed carbohydrate attachment site mutant of clone 8.3, so that the product of the mutant would lack the amino terminus proximal carbohydrate attachment site (CHO1). The mutation changed the asparagine residue the Asn-Trp-Ser sequence at CHOI site to a glutamine residue. However, transfection of COS7 cells with the mutant resulted in the formation of both the 60-kd and 11-kd IgE-PF which had affinity for lentil lectin, indicating that the 11-kd IgE-PF contains the amino-terminusdistal glycosylation (CHO~)site. On the other hand, evidence was obtained that the ll-kd IgE-PF does not contain the CHO1site (28). However, the 11-kd factor bound to antibodies against a synthetic peptide corresponding to a segment between the two. CHOsites, indicating that the l l-kd factor contains this segment(29). A DNAsegment near the C-terminus of the clone 8.3 has a striking homologywith a highly conserved region of the reverse transcriptases of several retroviruses (27). Indeed, the cDNA clone hybridized with a cloned mouseintracisternal A-particle (IAP) gene but not with DNAfrom several other cloned retroviruses (29). A comparison of the DNAsequence of the clone 8.3 and a partial sequence of the genomic IAP clone showed that these sequences share extensive homologythroughout the region of the cDNAclone from which the 11-kd IgE-bF is derived. Furthermore, rabbit antiserum against electrophoretically isolated IAP structural protein gp 73 absorbed not only IgE-bF derived from the cDNAclone 8.3 but also those produced either by the hybridoma 23B6 or MLN cells of Nb-infected rats. However,neither IAP nor gp 73 is released from the cells. Manycells transcribe IAP genes abundantly but do not express detectable IgEobF or Fc~R. In addition, only a small number(4 out of 70) of cross-hybridizing cDNA clones from the 23B6 library express IgE-bF activity (27). Gene cloning of rodent IgE-bF suggested possible relationships among the 60-kd, 30-kd and 15-kd IgE-bF from the T cell hybridoma 23B6 and those from murine T cells (20). Since the 60-kd IgE-bF molecule composed of a single polypeptide chain, the 30-kd and 15-kd IgE-bF should be posttranslational cleavage products of the 60-kd precursor molecules. A fraction of the 60-kd IgE-bF was cleaved by reduction and alkylation treatment to yield the 30-kd, 15-kd, and 10-kd IgE-binding fragments, and the same treatment of the 30-kd IgE-bF yielded the 15-kd and 10-kd fragments. Reduction and alkylation of the recombinant 60-kd IgE-PF and cDNAclone 8.3 also yielded the 11-kd IgE-PF. It appears that a fraction of the 60-kd peptide was already cleaved by proteolytic enzyme(s), but the fragments were held together by intrachain disulfide

Annual Reviews 518 ISHIZAKA bonds. Since the predicted amino acid sequence of the recombinant 60-kd peptide contains 9 cysteine residues, it was anticipated that someof these residues were involved in intrachain disulfide bonds.

Annu. Rev. Immunol. 1988.6:513-534. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

ROLE OF CARBOHYDRATE MOIETIES IN IgE-bF MOLECULES IN THEIR BIOLOGIC ACTIVITIES Differences between15-kd IgE-PFand IgE-SFin their affinities for various lectins suggested a possible role of carbohydrate moieties in their biologic activities. The hypothesis was supported by the fact that rat T cells activated by 10/tg/ml Con A, which produced IgE-PF upon incubation with IgE, produced IgE-bF with suppressive activity when they were incubated with IgE in the presence of tunicamycin which inhibits the assembly of Nlinked oligosaccharides (30). Pretreatment of the same Con A-activated cells with glucocorticoids followed by incubation with IgE, or incubation of the cells with IgE in the presence of lipocortin, a phospholipaseinhibitory protein, induced the formation of IgE-SF which had affinity for PNA(3 l, 32). Furthermore, the T cell hybridoma clone 23B6, which produces IgESF upon incubation with IgE, formed IgE-PF when they were incubated with IgE in the presence of monoclonal antilipocortin antibody, which activated phospholipase(33). Sucha switching of the biologic activities IgE-bF was observed in humanT cell hybridoma as well (22). Whenthe hybridoma 166A2 were incubated with homologous IgE, essentially all IgE-bF formed by the cells had affinity for Con A. However,only a small fraction of the factors had affinity for lentil lectin, and the factors exerted only weak potentiating activity on the IgE response. If the same cells were incubated with IgE in the presence of bradykinin, which activates phospholipase, essentially all IgE-bF formed by the cells had affinity for lentil lectin. The factors had muchhigher potentiating activity than those formed in the absence of bradykinin. In contrast, incubation of the hybridomacells with IgE in the presence of lipocortin resulted in the formation of IgE-bF that had affinity for PNA(but not for Con A), and these factors selectively suppressed the lgE response. The capacities of a T cell clone to form either IgE-PF or IgE-SF under different conditions suggested that IgE-PF and IgE-SF are structurally related. The hypothesis was supported by transfection of COS7 cells with a single cDNA clone. The transfection of the cells with the cDNA clone 8.3 resulted in the formation of both the 60-kd and 11-kd IgE-PF. However,if the transfection was carried out in the presence of tunicamycin, IgE-bF formed by the cells lacked affinity for lentil lectin and Con A, and the factors suppressed the IgE response (28). The results suggest that IgE-PF

Annual Reviews

Annu. Rev. Immunol. 1988.6:513-534. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

IgE-BINDING FACTORS

519

and IgE-SF mayshare a commonstructural gene, therefore a common polypeptidechain, and that biologic activities of IgE-bFare determined by posttranslational glycosylationprocess. Theeffect of tunicamycinon the biologic activities of IgE-bFindicate that N-linked, mannose-richoligosaccharide(s) in IgE-PFmolecules essential for their biologicactivities. As describedabove,the recombinant 60-kd IgE-bF of the carbohydrate attachment site mutant of cDNA clone 8.3 lacks an N-linked oligosaccharideattached to the CHO1 site, but it contained an N-linked oligosaccharide attached to the CHO2 site and exertedpotentiating activity on the IgEresponse(28). This result suggests that N-linkedoligosaccharideattached to the CHO2 site is essential for IgE-potentiatingactivity. Structures of oligosaccharides in IgE-PFand IgE-SFmolecules are unknown.However, the 15-kd rat IgE-PF have both N-linked oligosaccharide and O-linkedoligosaccharide(s), and their terminal residues are sialic acids (26). Thebiologic activities of the 15-kdIgE-PFwerelost by treatment of the factors with neuraminidase(34), indicating that the terminalsialic acid residuesare essential for the potentiating activity. On the other hand, the 15-kd IgE-SF appears to contain O-linked oligosaccharides whoseterminal residues are galactose ~ N-acetylgalactosamine(26). Althoughthis factor doesnot haveaffinity for either ConA lentil lectin, it is not clear whether the factor contains an N-linked oligosaccharidewith no affinity for the lectins or lacks such an oligosaccharide. Nevertheless, N-linked oligosaccharide does not appear to be involved in the function of IgE-SF, becausethe presence of tunicamycindoes not affect the biologic activities of IgE-SFformedby T cells (26). Fc~RII ON B LYMPHOCYTES AND B CELLDERIVED IgE-bF Since IgE-bFhaveaffinity for IgE, it wasoriginally anticipated that the factors werederivedfromFc,Ron lymphocytes (34). It waswell established + cells are B cells in both humanand rodent (35, that the majority of Fc~R 36), and that B lymphoblastoidcells transformedby EBvirus bear a high density of Fc, R. Kikutaniet al (37) reported that essentially all mature #+6+ humanB lymphocytes bear Fc~R, as determined by fluorescent staining with monoclonalanti-Fc, R antibody, while other subsets of B cells bearing surface IgG, IgA, or IgE do not express Fc,R. TheFeaRon mouseB cells and humanlymphoblastoid RPMI8866 cells represent a single polypeptidechain of a molecularweightof 49 kd and 47 kd, respec-

Annual Reviews

Annu. Rev. Immunol. 1988.6:513-534. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

520 ISHIZAKA tively (38). Sarfati et al (39) actually have shownthat culture supernatants of humanB lymphoblastoid RPM18866 cells contained soluble substances that inhibited rosette formation of Fc, R+ cells with IgE-coated erythrocytes. The major component of this factor had a molecular weight of 25 ~ 29 kd, bound to monoclonal antibody against Fc~R, and was proved to be a fragmentof Fc, R (40, 41). Structures of Fc~R on RPMI8866 cells and IgE-bF derived from the cells becameclear by cloning of genes for the receptors, by three groups of investigators (41-43). The receptor molecule is a single glycopeptide chain consisting of 321 aminoacids. The hydrophilicity plot of the peptide indicates the lack of an amino terminal signal sequence and the presence of a putative transmembraneportion near the amino terminal end (aminoacid residues 35 ,-~ 45). The findings suggest that the carboxy terminus is exposed to the cell exterior, and the amino terminus is cytoplasmic. The sequence of the peptide has homologywith animal lectins such as chicken hepatic lectin, humanand rat asialoglycoprotein receptor, and rat mannose-binding protein C, of which the amino terminus is located on a cytoplasmic slide (41, 42). The nucleotide sequence of Fc~Rgene shares no homologywith that of rodent T cell-derived IgE-bF described above, nor with Fc~Ron lymphocytes (44) or Fc, RI on rat basophilic leukemia cells (45). Partial amino-acid sequencing of the 25-kd soluble fragment indicates that the fragment with affnity for IgE represents the carboxy terminal half, i.e. amino acid 148-321, of the receptor peptide (41, 42). Thus, the fragment appears to be a proteolytic cleavage product of Fc, RII. The predicted aminoacid sequence of Fc, RII peptide also indicated that the receptors on humanB cells have only one N-linked glycosylation site, and that the 25-kd IgE-binding fragment does not contain any N-linked glycosylation site (42, 46). The t’ragment was detected in the s~rum both normal individuals and atopic patients, by radioimmunoassay using monoclonal anti-Fc,RII antibody (46). However, lack of an N-glycosylation site in the 25-kd fragment indicates that the soluble fragment of the Fc, R is distinct from T cell-derived IgE-PF, which have affinity for both ConA and lentil lectin. It is knownthat Fc~RIIon mouseB cells also degrades at the cell surface by means of proteolytic enzymeinto a 38-kd soluble fragment and a 10kd fragment the latter of which remains associated with the cell membrane (47). The 38 kd soluble fragment from mouse B cells binds to monoclonal anti-Fc~R antibody. However, in contrast to the humanFc, RII fragment, the soluble fragment from mouseFc~Rdoes not have affinity for IgE (47). This finding is in agreement with previous observations that rodent IgEbF is derived from T cells rather than B c~lls (1).

Annual Reviews

Annu. Rev. Immunol. 1988.6:513-534. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

IgE-BINDING

FACTORS 521

In humans,Fc~RIIis expressednot only on B cells but also on macrophages (48), platelets (49), and activated eosinophils (50). However,Kikutani al (42) could not detect Fc~RmRNA in the T cell line CEMnor normalT lymphocytesby Northern blot analysis. The receptor mRNA was expressed only in HTLV-Itransformed T cell line. Immunofluorescence staining of peripheral blood lymphocytes with monoclonal antibody against B cell Fc~RIIfailed to demonstratethe receptors on normalT cells (37). However,Younget al (24) established human+ T cell clones from peripheral blood lymphocytesof atopic dermatitis. Prinz et al (51) reported that a subset of activated humanT lymphocytesof atopic patients ÷ formedrosettes with red cells coated with monoclonalanti-Fc~R. Fc~R T cells werealso demonstratedby rosetting techniquein MLN cells of Nbinfected rats (52). Both rodent T cell hybridomas(53) and humanT hybridomas(54) respondedto homologousIgE or to antibodies reacting to Fc~Rfor the formationof IgE-bF.Thesefindings suggest that a subset of T cells from both humanand rodents probably comprise a minimum number of cell surfacereceptors for IgE, whichis not sufficient for detection either by rosetting techniques or by immunofluorescence.Since a monoclonalantibody against humanIgE-bF, which did not react with Fc~RIIon B cells, stimulated humanT cell hybridomasfor the production of IgE-bF(54), it is quite possible that the receptors on T cells may structurally different fromFc~RIIon B cells. Thebiological role of Fc~RIIon B cells is not known.Since the receptor is expressedon resting B cells but not on sIgE+ B cells that are committed for IgEsynthesis(37), it is rather unlikelythat the receptorsplayregulatory roles exclusively for the IgE response. Nevertheless, Sarfati et al (39) reported that the soluble fragments of Fc~RII on RPMI8866 cells enhancedIgE synthesis of humanlymphocytes.An unexpectedfinding is that Fc~RIIon humanB cells is identical with a B cell differentiation antigen knownas CD23 or Blast 2 (55, 56), whichis especially prominent after EBvirus infection on B cells (57). Gordon et al (58) previouslyfound that in the presenceof phorbolester, a monoclonalantibodyagainst CD23 induces the progressionof B cells throughthe GI phase of the cell cycle. The antibody also enhancesthe release of the CD23fragment, i.e. 25-kd Fc~RII fragment, from B cells (59), whichmaybe an autocrine B cell growth factor (60). Swendman and Thorley-Lawson(60) suggested intact CD23/Fc~RIIis a membraneform of IL-1. Since the monoclonal anti-CD23has proliferative effects on B cells, similar to low molecular weight BCGF,CD23 (Fc~RII) maybe a receptor for low molecularweight BCGF (61). Althoughthe problemis not yet settled, Fc~RII on B cells and/or the 25-kd soluble fragmentof the receptor moleculeappear to be involved in B cell differentiation and/or proliferation and mayplay an

Annual Reviews

522 IS~ZAr~A importantrole in cell transformation,rather than in the isotype-specific regulation of IgEresponses. MECHANISM FOR THE FORMATION

Annu. Rev. Immunol. 1988.6:513-534. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Lymphokines for Induction

OF IgE-bF

of lgE-bF Formation

Analysisof cellular mechanisms for the selective formationof either IgEPF or IgE-SF by rodent lymphocytes under various experimental conditions showedthat in all systemsstudied, the major cell sourcesof IgEbF are Lyt 1 ÷ T.cells whichbear either Fc~Ror F%Ror both. Whenrats and mice were treated with CFAor Bordetella pertussis vaccine, their macrophagesand/or monocyteswere activated, and then they released TypeI interferon, whichin turn stimulated FcR+ T cells to formIgE-bF (62, 63). Purified mouse/~interferon inducednormallymphocytesfor the formation of IgE-bF (15). Whenanimals were primed with a protein antigen and their spleen cells stimulatedwith the homologous antigen, Lyt 1 ÷ antigen-primed helper T cells released T cell factor(s) whichstimulated unprimedFcR+T cells to form IgE-bF(64). Recent experimentsrevealed that recombinantmouse~ interferon (but noneoflL-1, IL-2, IL-3 and IL4) induced normalBALB/csplenocytes for the formation of IgE-bF. IL-4 was shownto induce biosynthesis of Fc~Rin B cells and enhanced the expressionof the receptors on their surface (65, 66). In the rodent system,incubationof B cells with 3 ,~ 5 U/mlof IL-4 increasedthe density of Fc~RIIon the majority of B cells and enhancedthe release of 38-kd fragmentsof the receptors whichlacked affinity for IgE. It has beenshown that mouseIgE also induced an increase in Fc, R+ cells in mouseMLN cells, as determinedby rosetting techniques. Lee et al (47) have shown + B cell hybridomas that IgE prevents the degradation of Fc, R on Fc~R and that this mechanism is responsible for an increase in the density of Fc, R on their surface. Thus, the combinationof IL-4 and IgE markedly increases the density of Fc, R on B cells. HumanIL-4 also induces an increase in Fc~Ron human peripheral bloodB cells, particularly if the cells are activated by anti-/~ chain antibodies(66), and enhancesthe release the 25-kdfragmentof Fc~R,whichhas an affinity for IgE. Thus, in human systems,different lymphokines,i.e. interferon and IL-4, appear to induce the formationofT cell-derived IgE-bFand B cell-derived IgE-bF,respectively. It is interesting that the effects of IL-4 on B cells to enhancethe expressionof Fc,R and Ia moleculesare prevented by IFNyin both rodent and humansystems (66, 67). This principle, however,maynot apply for the other cells. In humanmacrophagescell line U937cells, as well as Burkitt virus-transformed B cells, JIJOY,both IL-4 and IFN~increase mRNA for Fc~RII (J. ¥odoi, personal communication).

Annual Reviews

Annu. Rev. Immunol. 1988.6:513-534. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

IgE-BINDING FACTORS

523

Lymphokines Controlling the Biologic Activities of IgE-PF or IgE-SF WhenBDF1mousespleen cells were stimulated with "inducer" of IgEbF (or interferon), IgE-PFwas formed. Stimulation of SJLmousespleen cells with the same"inducer" resulted in the formation of IgE-SF(9). Thus,interferons do not determinethe biologic activities oflgE-bF.Under ÷ physiologicalconditions, biologic activities of the factors formedby FcR T cells are controlled by twoT cell factors, i.e. glycosylationenhancing factor (GEF)and glycosylationinhibiting factor (GIF), whichregulate posttranslational glycosylation process of IgE-bFpeptide (4). Glycosylation enhancingfactor (GEF)is derived froma subset of Lyt ÷ T cells. Whenanimals are treated with pertussis vaccine for the selective formation of IgE-PF, pertussis toxin stimulates Lyt 1 ÷ T cells to form GEF(62, 68). Immunization of Lewisstrain rats or BDF1 mice with alumabsorbedantigen results in primingof not only helper T cells but also Lyt 1÷, FcR÷ T cells, and this latter subset releases GEFupon antigenic stimulation (64). Thus, whenunprimedFcR÷ T cells are stimulated by inducers (interferon) in the presenceof GEF,these cells selectively form IgE-bFhavinga "proper"N-linkedoligosaccharide, whichpotentiates the IgE response(cf Figure2). Onthe other hand, the sameFcR÷ T cells selectively form IgE-SF,when the cells are stimulatedby inducers (interferon) or IgEin the presence GIF. WhenLewisrats are primedwith a protein antigen included in CFA, not only Lyt 1+ helper T cells but also Lyt 2+ T cells are primed.Thus,

LytI ¢

"inducer"or

Figure 2 Schematic models for the selective formation of IgE-potentiating factor or IgEsuppressive factor. FcR+ T cells form IgE-potentiating factor in the presence of GEF,but the same cells form IgE-suppressive factor in the presence of GIF.

Annual Reviews

Annu. Rev. Immunol. 1988.6:513-534. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

524 ISHIZAKA antigenic stimulation of spleen cells results in the formation of ~ interferon from helper T cells and GIF from Lyt 2+ T cells. These two factors in combination stimulate FcR+ T cells to form IgE-SF (64). Repeated injections of CFAalso stimulate Lyt 2+ T cells to form GIF (63). In the mouseLyt 2+, I-J + antigen-specific suppressor T cells formed GIF (11). GIF from lymphocytes of CFA-treated rats has a mol wt of 13,000 to 15,000 and binds to monoclonal antibody against lipocortin (32). The lymphokinedid not exert phospholipase A2 inhibitory activity by itself but inhibited phospholipase A2 after treatment with alkaline phosphatase. Thus, GIF is apparently a phosphorylated derivative of phospholipase inhibitory protein. Purified lipocordn from rabbit neutrophils (69) as well as recombinant humanlipocortin I (70), at the level of 0.1 #g/ml, could switch normal mouselymphocytes for the selective formation of IgE-SF. Treatment of normal mouse splenic lymphocytes with glucocorticoids induced Lyt 2+ T cells for the formation of GIF (71). An interesting observation was that GIF from mouse lymphocytes possessed I-J determinant(s); GIF from H-2b strains bound to anti-I-J b alloantibodies and to monoclonal anti-I-J b, while GIF from H-2k kstrains bound to anti-I-J antibodies (71). It is rather unlikely that GIFis a fragment of lipocortin. However, GIF shares commonbiochemical properties and an antigenic determinant with lipocortin. One may speculate that the phospholipase inhibitory activity of the lymphokineis important in its immunological functions. GEFwas inactivated by inhibitors of serine protease, was bound to p-aminobenzamidine agarose, and could be recovered by elution with benzamidine(72). Effects of various inhibitors of trypsin-like enzymes GEFsuggested that GEFwas a kallikrein-like enzyme. This speculation was supported by the fact that trypsin and kallikrein, as well as bradykinin (a cleavage product of kininogen by kallikrein), have GEFactivity and switch T cells for the selective formation of IgE-PF. However,.GEF has lectin-like properties and binds to acid-treated Sepharose (68). The effect of GEF, which would otherwise switch the nature of IgE-bF, was prevented by d-galactose, ~¢-lactose, and N-acetylgalactosamine;this indicates that GEFexerts its function by binding to d-galactose on the cell surface (73). GEFconsists of two molecular weight species of 45 ~ 55 kd and 25 kd as estimated by gel filtration (73). One of the unique properties of mouse GEFis that the lymphokine binds to alloantibodies against class II MHCantigens. GEFform H-2k mice bound to anti-Ia k alloantibodies, while GEFfrom H-2b mice bound to anti-Ia ~ alloantibodies (73). GIF and GEFcompete with each other with respect to the glycosylation process of IgE-binding peptide(s). These two lymphokines do not interact with each other, but a mixture of GEFand GIF fails to change the nature

Annual Reviews

Annu. Rev. Immunol. 1988.6:513-534. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

IgE-BINDING FACTORS

525

of IgE-bFformedby T cells (74). Underphysiological conditions, the balance between GEFand GIF appears to determine the nature of the IgE-bF formed. Table 1 summarizesvarious systems in which the IgE antibody responseis either enhancedor suppressed. TheIgE-PFis always detected whenIgE responseis enhanced.In these systems, GEFis detected in culture supernatants of lymphocytes.Onthe other hand, formation of IgE-SFis always accompaniedby the formation of GIF(75). It is not knownwhyGEFand GIF competewith each other in terms of the glycosylationof IgE-bF.Biochemicalanalysis of the effect of GEF on a T cell hybridoma23A4cells revealed that GEFactivated membraneassociated enzymessuch as methyltransferases and phospholipase C, induced Ca-influx and the formation of diacylglycerol, whichin turn activated Ca-activated, phospholipid-dependentprotein kinase (protein kinase C) (76). GEFalso induced the release of arachidonate from cells (74). GEFappears to induce the activation of phospholipaseA2and phospholipaseC in the T cells. Since GIFis a derivative of phospholipase inhibitory protein, antagonistic effects betweenGEFand GIFwith respect to the glycosylationof IgE-bFmaybe related to activation vs inactivation of phospholipasein the cells by the two lymphokines. Formation of IgE-bF by Antigen-Specific T Cells Induction of IgE-PF or IgE-SF formation by lymphokines, described above(cf Figure 2), does not exclude the possibility that someof the antigen-specific T cells mayform IgE-bF. Indeed, we have obtained an antigen-specific mouseT cell clone andantigen-specific T cell hybridomas whichproduce IgE-bF uponincubation with OA-pulsedsyngeneic macroTalfle 1 Correlation amongthe IgE response, nature of lgE-bF, and modulators of glycosylation Experimental procedures

IgE response

IgE-bF

Modulators of glycosylation

Nb-infection, 2 weeks BP-treatment aKLH+ alum priming bBDFImice priming

Enhancement Enhancement IgE-Abresponse IgE-Abresponse

Potentiating Potentiating Potentiating Potentiating

GEF GEF GEF GEF

CFA-treatment ~ KLH+ CFApriming bSJL mice priming 23B6T cell hybridoma

Suppression No IgE-Abresponse NoIgE-Ab response

Suppressive Suppressive Suppressive Suppressive

GIF GIF GIF GIF

Lewisstrain rats wereprimedwithKLH. Either BDF1 or SJLmicewereimmunized with alum-absorbed OA.

Annual Reviews

Annu. Rev. Immunol. 1988.6:513-534. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

526

~SmZA~A

phages. It was found that someof antigen-specific T cell hybridomassuch as 231F 1 constitutively formed GIF and produced both IgE-SF and IgGsuppressive factors upon incubation with OA-pulsed macrophages (21). Wehave also obtained the mouseT cell hybridomas 12H5, which produced IgE-PF and IgG-potentiating factors upon antigenic stimulation (77). expected, this type of hybridomaconstitutively secrete GEF. The response of these T cell hybridomasto the antigen is MHC-restricted. The T cell hybridoma 231F 1 cells respond to OA-pulsedmacrophages of BDF1or BALB/cmice for the production of IgE-SF, but not to OApulsed macrophagesof H-2k or H-2b strains nor to free OA.The hybridoma 12H5 cells respond to OA-pulsed macrophages of BDF1or H-2b strain for the production of IgE-PF, but not to OA-pulsed macrophages of H2b or H-2k strains. Pretreatment of antigen-presenting cells with monoclonal anti-lA d antibody prevented antigen presentation to 231F1 cells for IgE-bF production. Similarly, treatment of antigen-presenting cells with anti-IAb, but not with anti-IA~, alloantibodies prevented the antigeninduced formation of IgE-bF by 12H5cells. The antigen recognized by these hybridomas does not appear to be the native molecules. Syngeneic macrophagesor A20.3 cells (78), which were pulsed with Urea-denatured OAor tryptic digests of the protein, stimulated 231F1 cells for the formation of IgE-bF. The results collectively suggest that these antigenspecific T cell hybridomasbear T cell receptors that recognize a fragment of antigen associated with Ia molecules on the antigen-presenting cells. The results also imply the presence of a subset of antigen-specific "regulatory" T cells which form IgE-bF and IgG-bF upon antigenic stimulation. Since the same hybridomas respond to mouse IgE for the formation of IgE-bF, these T cells should bear a minimumnumber of Fc~R(and F%R). Whena suspension of antigen-primed spleen cells were incubated with homologous antigen, unprimed FcR÷ T cells stimulated by lymphokines (Figure 2) maybe the major source of IgE-PF or IgE-SF. However, it conceivable that cognate interaction between the antigen-specific regulatory T cells and the antigen-presenting cells in lymphoidtissues mayplay an important role in the regulation of the IgE antibody formation in the tissues. An important question remaining to be answered is: What kind of T cell subset is represented by the hybridomas? Incubation of 231F1 cells with OA-pulsedsyngeneic macrophagesresulted in the formation not only of IgE-SF but also of GIF which has affinity for OA(79). Similarly, the 12H5 cells formed GEFthat have an affinity for OA, when they were incubated with OA-pulsed syngeneic macrophages (77). The OA-specific GIF from 231F 1 cells and OA-specific GEFfrom 12H5cells lacked affinity for KLHor BSA, which suggests the antigen-specificity. Whenthese

Annual Reviews

Annu. Rev. Immunol. 1988.6:513-534. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

IgE-BINDING

FACTORS

527

hybridomas were cultured without antigenic stimulation, the cells constitutively released either GIF or GEF,both of which lacked affinity for OA. The OA-specific GIF was also released from spleen cells of BDF1 mice which received repeated i.v. injections of OAfor the generation of antigen-specific suppressor T cells (11). Onthe other hand, spleen cells BDF1 mice primed with alum-absorbed OA formed OA-specific GEF upon antigenic stimulation (73). Evidence has accumulated that antigenspecific GIF is similar to antigen-specific TsF described by manyinvestigators. Intravenous injections of an appropriate dose of OA-specific GIF from 231F1 cells suppressed both IgE and IgG anti-DNP antibody responses of BDF1 mice to alum-absorbed DNP-OA but failed to suppress the anti-DNP antibody responses of the same strain to DNP-KLH (79). The OA-specific GIF consisted of two molecular weight species of approximately 80 kd and 35 -,, 40 kd, respectively, as estimated by gel filtration, while nonspecific GIF from the same cells was 15 kd. Reduction and alkylation treatment of the 40 kd OA-specific GIF yielded the 15 kd nonspecific GIF (80). Although the molecular characterization of OAspecific GIFrequires further studies, the antigen-specific T cell factors appear to be composedof multiple polypeptide chains. One chain has an affinity for antigen and another chain has I-J determinant, binds to antilipocortin, and has GIFactivity (79). Thus, antigen-specific GIFis similar to GAT-specific TsF2 (81), KLH-specific TsF (82), and NP-specific (83) in their immunosuppressive effects, in antigenic structures, and in the capacity to bind antigen. On the other hand, antigen-specific GEFfrom 12H5cells are similar to the antigen-specific T cell augmenting factor (TaF) described by Tokuhisaet al (84) and analyzed by Miyatani et al (85). Thus, OA-specific GEFenhances in vitro anti-DNP antibody response by DNP-OA-primedcells to homologous antigen, while KLH-specific GEF enhances the anti-DNP antibody response of DNP-KLH-primed cells to DNP-KLH (73). Both the antigen-specific TaF and antigen-specific GEF failed to replace antigen-specific helper T cells in the secondary antibody response, and both factors bind to alloantibodies against Ia. Indeed, GEF from 12H5cells bound to anti-I-A b, but not to anti-IAd or anti-IA k alloantibodies. However, it is doubtful that GEF from the hybridoma is a product of I-Ab subregion. Tokuhisa et al (84), as well as Hiramatsu et al (86), reported that the I-A specificities associated with TaF were serologically distinct from those of B cell-associated Ia molecules, and unique to the T cells (Iat) (87). The antigen-specific GEFfrom 12H5cells consisted of two molecular weight species of 80 kd and 55 kd, respectively, as estimated by gel filtration; and both species bound to OASepharose. Recent experiments suggest that antigen-specific GEFare probably composed of antigen-specific chain and nonspecific GEF

Annual Reviews

Annu. Rev. Immunol. 1988.6:513-534. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

528

ISHIZAKA

(which have the molecularweight of either 55 kd or 25 kd and of Ia(t) determinant). One mayspeculate that the hybridoma231F1represents a subset of antigen-specific suppressorT cells whichform not only antigen-specific TsF(GIF) but also isotype specific Ig-binding suppressive factors upon antigenic stimulation. On the other hand, 12H5cells mayrepresent a subset of "amplifier" T cells, whichproduceantigen-specific TaF(GEF) and isotype-specific, Ig-binding potentiating factors. Weanticipate that suchsuppressorT cells as well as amplifier T cells mayparticipate in the regulation of the antibodyresponses throughthe formationof Ig-binding factors andantigen-specificT cell factors. POSSIBLE APPROACHES TO SUPPRESS THE IgE ANTIBODY RESPONSE BY ANTIGEN-SPECIFIC GIF Properties and cell source of GIFsuggested that nonspecific GIFmay also have immunosuppressive activity. Indeed, repeated injections of a relatively large quantity of the nonspecific, 15-kdGIFfrom23A4cells to BDF1mice during the primary response to alum-absorbed DNP-OA completely suppressed both IgE and IgG-antibody formation (88). The same GIF treatment of BDF1mice primed with alum-absorbed OA switched their spleen cells from the formation of IgE-PFto the formation of IgE-SF. As described, stimulation of spleen cells of the OAprimed BDF1mice with OAresulted in the formation of IgE-PF and GEF(9). In contrast, spleen cells of OA-primed, GIF-treated mice formed IgE-SFand GIFuponantigenic stimulation. The GIFformedby the latter spleen cells had affinity for OAand boundto anti-I-J b alloantibodies. ÷, Furthermore, spleen cells of the GIF-treated mice contained Lyt 2 OA-specific suppressor T cells, which released OA-specific GIFupon antigenic stimulation (88). Thus, GIF treatment of immunizedmice appears to facilitate the generation of antigen-specific suppressor T cells in vivo, and these cells maybe responsible for the suppression of the antibody response. Basedon the findings described above, attempts were madeto generate antigen-specific suppressorT cells in vitro (cf Figure3). BDF1 micewere primedwith alum-absorbedOAfor a persistent IgE antibody formation, and their spleen cells werestimulated with OAto activate antigen-primed T cells. Theactivated T cells werethen propagatedwith IL-2 in the presence or absence of nonspecific GIF. The cells propagated with IL-2 alone formed IgE-PF and antigen-specific GEFupon stimulation with OApulsed syngeneicmacrophages.In contrast, the cells propagatedwith IL2 in the presence of GIFformedIgE-SF and antigen-specific GIFupon

Annual Reviews IgE-BINDING

alum-absorbed

Activated

OA --

T "

(spleen

cells

T cells

. cells~ ~T

+ OA)

cells

+

529

ture

+ OA-M~ 24 OA-.

24

Figure 3 Experimental protocol for the generation of antigen-specific that produce antigen-specific GIF upon antigenic stimulation.

Annu. Rev. Immunol. 1988.6:513-534. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

FACTORS

h~r hr

~ ~

suppressor

T cells

antigenicstimulation(89). Toobtain a sufficient amountof antigen-specific GIF, OA-primed T cells stimulated by the antigen were propagatedby IL2 in the presence of nonspecific GIF, and these cells were fused with BW 5147. Morethan one-half of T cell hybridomasobtained constitutively secreted GIF, and about one-half of the GIF-producing hybridomas formed OA-specific GIF upon incubation with OA-pulsed syngeneic macrophages.Injections of the OA-specificGIFobtained from a representative T cell hybridomasuppressed both IgE and IgG antibody responses of BDF1mice to DNP-OA,but the same treatment failed to suppress the antihapten antibody response of the same strain to DNPKLH,indicating that OA-specificGIFsuppressed the antibody response in a carrier-specific manner. SUMMARY In rodents, IgE-bFare derived froma subset of T cells that bear Fc~Ror F%R,or both, and selectively enhanceor suppressthe IgE response. IgEPF and IgE-SFmayshare a common structural gene, therefore a common polypeptide chain, and their biologic activities are decided by posttranslational glycosylationprocess. Underphysiologicalconditions, this process is controlled by two lymphokines,i.e. GEFand GIF. The same principle probablyapplies to humanT cell-derived IgE-bF.In both rodent and humanlymphocytes,Fc~RIIon B cells are degraded, and their fragments are released from the cells. The fragmentsof Fc~RIIon humanB cells represent the carboxyterminal half of the receptor moleculesand haveaffinity for IgE. In contrast, the fragmentof Fc~Rin mouseB cells does not have an affinity for IgE. Thus, "IgE-bF"are derived from both T cells and B cells in humans,but only from T cells in rodents. The formation of T cell-derived IgE-bFwas induced by interferons, while biosynthesis of Fc~Rin B cells and the formationof their fragmentswere enhancedby IL-4. IgE-bF are also formed by a subset of antigen-primed T cells upon

Annual Reviews

Annu. Rev. Immunol. 1988.6:513-534. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

530

ISHIZAKA

cognate interaction with antigen-pulsed syngeneic macrophages. These antigen-primed T cells constitutively secrete either GEFor GIF, having no affinity for homologousantigen. Uponantigenic stimulation, however, GEFand GIFformed by the cells had affinity for the antigen. The antigenspecific GEFenhanced the antibody response, and antigen-specific GIF suppressed the antibody response, both in carrier specific manner. The possible relationship between antigen-specific GEFand antigen-specific TaF, and that between antigen-specific GIF and antigen-specific TsF both require further studies. Nonspecific GIF not only switches T cells from the formation of IgEPF to the formation of IgE-SF, it also facilitates the generation of antigenspecific suppressor T cells which produce antigen-specific GIF upon antigenie stimulation. Propagation of antigen-primed T cells in the presence of GIFalso facilitate the generation of antigen-specific suppressor T cells in vitro. If the same procedures would be effective for humanT cells of allergic patients, it would be possible to generate antigen-specific suppressor T cells from their T cell population in vitro and to establish T cell hybridomas that produce allergen-specific GIF(TsF). Elucidation of the mechanisms for the generation of suppressor T cells by GIF requires further study, however, the method may provide a new approach to regulate the IgE antibody response in atopic patients. ACKNOWLEDGMENTS This work was supported by research grants AI-11202 and AI-14784 from the US Humanand Health Service and PCM-8100080from the National Science Foundation. The author expresses great appreciation to collaborators, Drs. M. Iwata, T. Uede, T. F. Huff, P. Jardieu, and M. Akasaki from the Johns Hopkins University, School of Medicine; and Drs. K. Moore and C. Martens from DNAX Institute of Molecular and Cellular Biology. LiteratureCited 1. Yodoi, J., Ishizaka, K. 1980. LymphocytesbearingFc receptorsfor IgE. IV. Formation of IgE-binding factor by rat T lymphocytes.J. lmmunol.124: 1322-29 2. Suemura, M.,Yodoi,J., Hirashima,M., Ishizaka, K. 1980.Regulatoryrole of IgE-bindingfactors from rat T lymphocytes.I. Mechanism of enhancement of IgEresponsebyIgE-potentiating factor. J. Immunol. 125:148-54 3. Hirashima, M.,Yodoi,J., Ishizaka,K. 1980.Regulatoryrole of IgE-binding

factorsfromrat Tlymphocytes. III. IgEspecific suppressivefactor with IgEbindingactivity. J. lmmunol. 125:144248 Ishizaka, K. 1984.Regulationof IgE synthesis.Ann.Rev. Immunol. 2:159-82 Hirashima, M.,Yodoi,J., Ishizaka,K. 1981.Regulatoryrole of IgE-binding factors fromrat T lymphocytes. V.Formationof IgE-potentiatingfactor by T lymphocytes fromrats treated withBordetella pertussis vaccine.J. Immunol. 126:838-42

Annual Reviews

Annu. Rev. Immunol. 1988.6:513-534. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

IgE-BINDINGFACTORS 53 l 6. Tung,A. S., Chiorazzi, N., Katz, D. H. 1978. Regulation of IgE antibody production by serum molecules. I. Serum from complete Freund’s adjuvant-immunedonors suppresses irradiation-enhanced IgE production in low responder mouse strains. J. ImmunoL120: 2050-59 7. Hirashima,M., Yodoi,J., Ishizaka, K. 1980. Regulatory role of IgE-binding factors fromrat T lymphocytes. IV. Formationof IgE-binding factors in rats treated with complete Freund’s adjuvant. J. lmmunol.125:2154-60 8. Uede,T., Huff,T. F., Ishizaka, K. 1982. Formationof IgE-bindingfactors by rat T lymphocytes.V. Effect of adjuvantfor priming immunizationon the nature of IgE-bindingfactors formedby antigenic stimulation. J. Immunol.129:1384-90 9. Uede,T.,Ishizaka, K. 1984.IgE-binding factors from mouselymphocytes. II. Strain differencesin the nature of IgEbinding factor. J. lmrnunol.133:359-67 10. Takatsu,K, Ishizaka, K. 1976. Reaginic antibody formation in the mouse.VII. Induction of suppressorT cells for IgE and IgG antibody responses. J. Immunol. 116:1257-64 11. Jardieu, P., Uede,T., Ishizaka,K. 1984. lgE-binding factors from mouseT lymphocytes, III. Role of antigen-specific suppressor T cells in the formation of IgE-suppressivefactor. J. Immunol.133: 3266-73 12. Kishimoto,T., Hirai, Y., Suemura,M., Yamamura, Y. 1976. Regulationof antibody response in different immunoglobulinclass I selective suppressionof anti-DNP IgE-antibody response by preimmunizationof DNP-coupled mycobacterium. J. Immunol.117:394-404 13. Suemura,M., Kishimoto,T., Hirai, Y., Yamamura, Y. 1977. Regulationof antibodyresponses in different inanaunoglobulin classes III. In vitro demonstration of IgE-class-specificsuppressor function of DNP-mycobacteriumprimedT cells andthe soluble factor released fromthese cells. J. Immunol.119: 159-65 14. Suemura,M., Shib.o, O., Deguchi,H., Yamamura, Y., Bottcher, I., Kishimoto, T. 1981. Characterizationandisolation of IgE class-specific suppressorfactor (IgE-TsF).I. Thepresenceof the binding sites for lgE and of the H-2geneproducts in IgE-TsF.J. Irnrnunol.127: 46571 15. Uede, T., Sandberg, K., Bloom,B. R., Ishizaka, K. 1983. IgE-bindingfactors from mouse T lymphocytes. I. Formationof IgE-bindingfactors by stimu-

lation with homologous IgE and interferon. J. ImmunoL 130:649-54 16. Chiorazzi, N., Fox, D. A., Katz, D. H. 1976. Hapten-specific IgE antibody responsein mice.VI. Selectiveenhancementof IgE-antibodyproductionby low dose X-irradiation and cyclophosphamide. J. Immunol.117:1629-37 17. Akasaki,M., Ishizaka, K. 1987. Effects of cyclophosphamide treatment and gammairradiation of SJL mice on the IgEantibody responseand the nature of IgE-bindingfactors. Int. Archs. Allergy Appl. IrnmunoL82:417-18 18. Saryan,J. A., Leung,D. Y., Geha,R. S. 1983. Induction of humanIgE synthesis by a factor fromT cells of patients with hyper IgEstates. J. Immunol.130: 24247 19. Leung, D. Y. M., Biozek, C., Frankel, R., Geha,R. S. 1984. IgE-specific suppressor factor in normalhumanserum. Clin. Immunol.Immunopathol.32: 33950 20. Jardieu, P., Moore,K., Martens, C., Ishizaka, K. 1985. Relationship among IgE-bindingfactors with various molecular weights. J. ImmunoL135:2727-34 21. Jardieu, P., Uede,T., Ishizaka,K. 1985. Presenceof an antigen-specific T cell subset that formsIgE-suppressivefactor andIgG-suppressivefactor on antigenic stimulation. J. Immunol.135:922-29 22. Huff, T. F., Jardieu, P., Ishizaka, K. 1986. Regulatoryeffect of humanIgEbinding factors on the IgE response of rat lymphocytes.J. Immunol.136: 95562 23. Huff,T. F., Uede,T., Ishizaka, K. 1982. Formationof rat IgE-bindingfactors by rat-mouseT cell hybridomas.J. Immunol. 129:509-14 24. Young,M. C., Leung, D. Y. M., Geha, R. S. 1984. Production of IgE-potentiating factor in manbyT cell lines bearing Fc receptors for IgE. Eur. J. Immunol. 14:871-78 25. Young,M. C., Geha, R. S., Maksad,K. N., Leung, D. Y. M. 1986. Characterization of humanT cell-derived IgEpotentiating factor. Eur. J. Immunol. 985-91 26. Yodoi,J., Hirashima,M., Ishizaka, K. 1982. Regulatory role of IgE-binding factors from rat T lymphocytes.V. The carbohydrate moieties in IgE-potentiating factors andIgE-suppressivefactors. J. Imrnunol.128:289-95 27. Martens, C, L., Huff, T. F., Jardieu, P., Trounstine, M.L., Coffman,R. L., Ishizaka, K., Moore,K. W.19~5. cDNA clones encoding IgE-binding factors from a rat-mouse T cell hybridoma.

Annual Reviews

Annu. Rev. Immunol. 1988.6:513-534. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

532

ISHIZAKA

Proc. Natl. Acad. Sci. USA82: 246064 28. Martens,C. L., Jardieu, P., Trounstein, M. L., Stuart, S. G., Ishizaka, K., Moore, K. W. 1987. Potentiating and suppressive IgE-binding factors are expressedby a single clonedgene. Proc. Natl. Acad. Sci. USA84:809-13 29. Moore,K. W., Jardieu, P., Mietz,J. A., Trounstine,M.L., Kuff, E. L., Ishizaka, K., Martens, C. L. 1986. Rodent IgEbinding factor genes are membersof an endogeneous J. Immunol.retrovirus-like 131:428390 genefamily. 30. Yodoi,J., Hirashima,M., Ishizaka, K. 198 I. Lymphocytes bearingFc receptors for IgE. V. Effect of tunicamycinon the formation of IgE-potentiating factor and IgE-suppressive factor by ConAactivated lymphocytes.J. ImmunoL 126: 877 82 31. Yodoi,J., Hirashima,M., Ishizaka, K. 198 I. Lymphocytes bearingFc receptors for IgE. VI. Suppressiveeffects of glucocorticoids on the expression of Fc, receptors and glycosylationof IgE-binding factors. J. Immunol.129:47176 32. Uede, T., Hirata, F., Hirashima, M., Ishizaka, K. 1983. Modulationof the biologic activities of IgE-bindingfactors. I. Identificationof glycosylation inhibiting factor as a fragmentof lipomodulin. J. Immunol.130:878-84 33. Huff, T. F., Uede, T., Iwata, M., Ishizaka, K. 1983. Modulationof the biologic activities of IgE-bindingfactors. III. Switchingof a T cell hybrid clone from the formation of IgE-suppressive factor to the formationof IgEpotentiating factor. J. Immunol.131: 1090-95 34. Yodoi,J., Hirashima,M., Ishizaka, K. 1980. Regulatory role of IgE-binding factors fromrat T lymphocytes.II. Glycoprotein nature and source of IgEpotentiating factor. J. Immunol.125: 1436-41 35. Gonzalez-Molina, A., Spiegelberg,H. L. 1976. Bindingof IgE-myelomaprotein to cultured lymphoblastoidcells. J. human Immunol. 117:1838-45 36. Fritche, R., Spiegelberg,H. L. 1978.Fc receptors for IgE on normalrat lymphocytes. J. lmmunol.121:471-76 37. Kikutani, H., Suemura,M., Owaki,H., Nakamura,H., Sato, R., Yamasaki,K., Barsumian,E. I., Hardy,R. R., Kishimoto,T. 1986. Fc~receptor, a specific differentiation markertransiently expressed on murine B cells before isotype switching. J. Exp. Med.164: 145569 38. Conrad, D. H., Peterson, L. H. 1984.

39.

40.

41.

42.

43.

44.

45.

46.

47.

The murine lymphocyte receptor for IgE. I. Isolation and characterizationof the murineB cell Fc, receptor and comparison with Fc, receptor from rat and humans. J. Immunol. 132:796803 Sarfati, M., Rector, E., Wong,K., Rubio-Trujillo, M., Sehon, A., Delespesse, G.1984.In vitro synthesisoflgE by humanlymphocytes. II. Enhancementof the spontaneousIgE synthesis by IgE-bindingfactor secreted by RPMI 8866lymphoblastoidB cell line. Immunology 53:197-205 Sarfati, M., Nakajima,T., Frost, H., Kilcher, E., Delespesse,G. 1987. Purification and partial biochemicalcharacterization of IgE-binding factors secreted from a human B lymphoblastoid cell line. Immunology 60:539-45 Ikuta, K., Takami, M., Kim, L. W., Honjo, T., Miyoshi, T., Tagaya, Y., Kawabe, T., Yodoi, J. 1987. Human lymphocyte Fc receptor for IgE: Sequencehomologyof its cloned cDNA with animal lectins. Proc. Natl. Acad. Sci. USA84:819-23 Kikutani, H., lnui, S., Sato, R., Barsumian,E. L., Owaki,H., Yamasaki,K., Kaisho, T., Uchibayashi, N., Hardy, R. P., Hirano,T., Tsumasawa, S., Sakiyama, F., Suemura,M., Kishimoto, T. 1986. Molecular structure of human lymphocyte receptor for immunoglobulin E. Cell 47:657-65 Ludin, C., Hofstetter, H., Sarfati, M., Levy, C. A., Suter, U., Alaimo, D., Kelchkerr, E., Frost, H., Delespesse, G. 1987. Cloningand expression of the cDNAcoding for a humanlymphocyte IgE receptor. EMBO J. 6:109-14 Ravetch, J. V., Luster, A. D., Weinshank, R., Kochar, J., Pavlorec, A., Portnoy, D. A., Hulmes, J., Pan, ¥. E., Unkeless,J. C. 1987.Structural heterogeneity and functional domainsof murine immunoglobulin G Fc receptors. Science 234:718-25 Kinet, J. P., Metzger,H., Kochar,J. 1987. cDNApresumptively coding for the ct subunit of the receptor of high affinity for immunoglobulinE. Biochemistry 26:460510 Sarfati, M., Nutman,T., Fonteya, C., Delespesse, G. 1986. Presenceof antigenic determinants commonto IgE Fc receptors on humanmacrophages,T and B lymphocytesand IgE-bindingfactors. Immunology59:569-75 Lee, W.T., Rao, M., Conrad, D. H. 1987. The murine lymphocytereceptor for IgE. IV. Themechanisms of ligandspecific receptorupregulationon Bcells. J. Immunol.139:1191-98

Annual Reviews

Annu. Rev. Immunol. 1988.6:513-534. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

IgE-BINDINGFACTORS 48. Melewitz, F. M., Spiegelberg, H. L. 1980. Fc receptors for IgE on a subpopulation of humanperipheral blood monocytes.J. lmmunol.125:1026-31 49. Joseph, M., Auriault, C., Capron, A., Vrong,H., Viens, P. 1983. A newfunction of platelets: IgEdependentkilling of Schistosomes.Nature303:819-12 50. Capron,M., Spiegelberg,H. L., Prin, L., Bennich,H., Butterworth,A. E., Pierce, R. J., Quaissi, M.A., Capron,A. 1984. RoleoflgEreceptorsin effector function of humaneosinophils. J. Immunol.132: 46248 51. Prinz, J. C., Endres,N., Rauk,G., Ring, J., Rieber, E. P. 1987.Expressionof Fc, receptors on activated humanT lymphocytes. Eur. J. Immunol.17:757-61 52. Yodoi, J., Ishizaka, K. 1979. Lymphocytes bearingreceptorsfor IgE. I. Presence of humanand rat T lymphocytes andFc receptors. J. Immunol.122: 257783 53. Huff, T. F., Yodoi, J., Uede, T., Ishizaka, K. 1984.Presenceof an antigenic determinant common to rat IgEpotentiatingfactor, IgE-suppressive factor and Fee receptors on T and B lymphocytes. J. Imrnunol.132:406-12 54. Kisaki, T., Huff, T. F., Conrad,D. H., Yodoi, J., Ishizaka, K. 1987. Monoclonal antibody specific for T cellderived humanIgE-bindingfactors. J. Immunol.138:3345-51 55. Yukawa,K., Kikutani, H., Owaki,H., Yamasaki,K., Yokota, A., Nakamura, H., Barsumian,E. L., Hardy,R. R., Suemura,M., Kishimoto,T. 1987,A B cellspecific differentiation antigen CD23 is a receptor for IgE (Fc~R)on lymphocytes. J. lmmunol.138:2476-80 56. Bonnefay,J.-Y., Aubry,J.-P., Peronne, C., Wijdenes,J., Banchereau,J. 1987. Production and characterization of a monoclonalantibody specific for the humanlymphocytelow affinity receptor for IgE: CD23 is a low affinity receptor for IgE. J. Immunol.138:2970-78 57. Thorley-Lawson,D. A., Nadler, L. M., Bhan, H. K., Schooley, R. T. 1985. Blast-2 (EBVCS), an early cell surface marker of humanB cell activation is super-inducedby EpsteinBarr virus. J. Imrnunol. 134:3007-12 58. Gordon,J., Rowe,M., Walker,L., Guy, G. 1986. Ligation of the CD23,p45 (Blast-2 EBVCS) antigen triggers the cell cycleprogressionof activated Blymphocytes. Eur. J. ImmunoL15:1075-80 59. Guy, G. R., Gordon, J. 1987. Coordinated action of IgE and a B cell stimulatory factor on the CD23receptor moleculeupregulates B lymphocyte

533

growth.Proc. Natl. Acad.Sci. USA84: 623943 S., Thorley-Lawson,D. A. 60. Swendeman, 1987. Theactivation antigen Blast-2, when shed is an autocrine BCGFfor normal and transformed B cells. EMBO J. 6:1637-42 61. Gordon,J., Webb,A. J., Walker, L., Guy,G. R., Row,M.1986. Evidencefor an association between CD23and the receptor for a low molecularweight B cell growthfactor. Eur.J. Immunol.16: 1627-30 62. Hirashima,M., Yodoi,J., Ishizaka, K. 1981. Formationof IgE-binding factor by rat T lymphocytes.II. Mechanisms of selective formationof IgE-potentiating factors by treatmentwithBordetellapertussis vaccine. J. Immunol.127:1804-10 63. Hirashima,M., Yodoi,J., Huff, T. F., Ishizaka, K. 1981. Formationof IgEbinding factors by rat T lymphocytes. III. Mechanisms of selective formation of IgE-suppressivefactors by treatment with complete Freund’s adjuvant. J. Irnmunol. 127:1810-16 64. Uede,T., Ishizaka, K. 1982. Formation of IgE-binding factors by rat T lymphocytes. VI. Cellular mechanismsfor the formationof IgE-potentiatingfactor and IgE-suppressivefactor by antigenic stimulation of antigen-primed spleen cells. J. Immunol.129:3191-97 65. Hudak,S. A., Gollnick, S. O., Conrad, D. H., Kethry, M. R. 1987. Murine B cell stimulatoryfactor 1 (interleukin 4) increases expression of the Fc receptor for IgE on mouseB cells. Proc. Natl. Acad. Sci. USA84:4606-10 66. Defrance, T., Aubry, J. P., Rousset, F., Vanbervliet, B., Bonnefoy,J. Y., Arai, N., Takebe,Y., Tokota,T., Lee, F., Arai, K., Vries, J., Banchereau,J. 1987. Humanrecombinant interleukin 4 induces Fc, receptors (CD23)on normal humanB lymphocytes.J. Exp. Med. 165:145947 67. Mond,J. J., Carman,J., Sarma, C., Ohara,J., Finkelman,F. D. 1986. Interferon-~,suppressesBcell stimulationfactor (BSF-I) induction of class II MHC determinantson Bcells. J. Immunol. 137: 3534-37 68. Iwata, M., Huff, T. F., Uede,T., Munoz, J. J., Ishizaka, K. 1983. Modulationof the biologicactivities oflgE-binding factor. II. Physicochemical properties and cell sources of glycosylationenhancing factor. J. Immunol.130:1802-8 69. Hirata, F., Schiffman, E., VenkataSubramanian,K., Solomon,D., Axelrod, J. 1980. A phospholipase A2 inhibitory protein in rabbit neutrophils in-

Annual Reviews

Annu. Rev. Immunol. 1988.6:513-534. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

534

ISHIZAKA

duced by glucocorticoids. Proc. Natl. Acad. Sci. USA77:2533-36 70. Wallner, B. P., Mattaliano, R. J., Hession, C., Cate, R. L., Tizand, R., Sinclair, L. K., Foeller, C., Chow, E. P., Browning,J. L., Ramachandran, K. L., Pipinsky, R. B. 1986. Cloningand expression of humanlipocortin, a phospholipase A2inhibitor with potential anti-inflammatoryactivity. Nature320: 77-81 71. Jardieu, P., Akasaki,M., Ishizaka, K. 1986. Association of I-J determinants with lipomodulin/macrocortin. Proc. Natl. Acad. Sci. USA83:160-64 72. Iwata, M., Munoz,J. J., Ishizaka, K. 1983.Modulation of the biologic activities of IgE-bindingfactors. IV. Identification of glycosylation enhancing factor as a kallikrein-like enzyme.J. Immunol. 131:1954-60 73. Iwata, M., Fukutomi, Y., Hashimoto, T., Sato, Y., Sato, H., Ishizaka, K.1987. Augmentationof the antibody response by antigen-specificglycosylationenhancing factor. J. Immunol.183:2561-67 74. Iwata, M., Akasaki, M., Ishizaka, K. 1984.Modulation of the biologic activities of IgE-bindingfactor. VI. Activation of phospholipase by glycosylation enhancingfactor. J. Immunol.133: 1505-12 75. Iwata, M., Huff, T. F., Ishizaka, K. 1984.Modulation of the biologic activities of IgE-bindingfactor. ¥. Therole of glycosylation enhancingfactor and glycosylationinhibiting factor in determining the nature of IgE-binding factors. J. Immunol.132:1286-93 76. Akasaki, M., Iwata, M., Ishizaka, K. 1985.Modulation of the biologic activities of IgE-bindingfactors. VII. Biochemical mechanismsby which glycosylation enhancingfactor activate phospholipase in lymphocytes.J. Immunol. 135:4069-77 77. Iwata, M., Adachi, M., Ishizaka, K. 1987.Antigen-specificT cells that form IgE-potentiating factor, IgG-potentiating factor and antigen-specific glycosylation enhancingfactor on antigenic stimulation. J. Immunol.In press 78. Glimcher,L. H., Kim,K.-J., Green,I., Paul, W.E. 1982.Ia antigen-bearingcell tumorlines can present protein antigen and alloantigen in a major histocompatibilitycomplexrestricted fashion to antigen-reactiveT cells. J. Exp.Med. 155:445-59 79. Jardieu, P., Akasaki,M., Ishizaka, K.

1987. Cartier-specific suppression of antibody responsesby antigen-specific glycosylation inhibiting factors. J. Immunol.138:1494-1501 80. Jardieu, P., Ishizaka, K. 1987. Possible relationship of an antigen-specificsuppressor factor to phospholipaseinhibitory protein. Immuneregulation by characterized polypeptide. UCLASymposium, Vol. 41, ed. G. Goldstein, J.F. Bach, H. Wigzell, pp. 595-604. New York: Alan R. Liss 81. Turck, C. W., Kapp,J. A., Webb,D. R. 1986. Structural analysis of a monoclonal heterodimeric suppressor factor specific for L-glutamicacid, -L alanineL tyrosine. J. lmmunol.137:1904-9 82. Saito, T., Taniguchi,M. 1984. Chemical features of an antigen-specific suppressor T cell factor composedof two polypeptidechains. J. Mol. Cell. Immunol. 1:137-45 83. Sherr, D. H., Minami,M., Okuda, K., Dorf, M. E. 1983. Analysis of T cell hybridomas.II. Distinctions between two types of hapten-specific suppressor factors that affect plaque forming cell response. J. Exp. Med.157:515-29 84. Tokuhisa, T., Taniguchi, M., Okumura, K., Tada,T. 1978. Anantigen-specificI region gene product that augmentsthe antibody response. J. lmmunol. 120: 414-21 85. Miyatani,S., Hiramatsu,K., Nakajima, P. B,, Owen,F. C., Tada,T. 1983.Structural analysisof antigen-specificIa-bearing regulatoryT cell factors: Gelelectrophoretic analysis of the antigen-specific augmentingT cell factor. Proc. Natl. Acad. Sci. USA80:6336-40 86. Hiramatsu,K., Miyatani, S., Kim,M., Yamada, S., Okumura, E., Tada, T. 1981. UniqueT cell Ia antigen expressed on a hybridcell line producingantigenspecific augmentingT cell factor. J. Immunol. 127:1118-22 87. Tada, T., Uracz,W., Abe, R., Arano,Y. 1985.I-J as an inducibleT cell receptor for self. Immunol.Rev. 83:105-24 88. Akasaki,M., Jardieu, P., Ishizaka, K. 1986. Immunosuppressive effects of glycosylation inhibiting factor on the IgE and IgGantibody response. J. Immunol. 136:3172-79 89. Iwata, M., Ishizaka, K. 1987. In vitro modulationof antigen-primedT cells by a glycosylation inhibiting factor that regulate the formation of antigen-specific suppressive factor. Proc. Natl. Acad. Sci. USA84:2444-48

Annu. Rev. Immunol. 1988.6:513-534. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Annu. Rev. Immunol. 1988.6:513-534. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Annual Reviews www.annualreviews.org/aronline Ann.Rev. Immunol.1988.6 : 535-54 Copyright© 1988by AnnualReviewsInc. All rights reserved

Annu. Rev. Immunol. 1988.6:535-554. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

NONP RECIPITATING ASYMMETRIC ANTIBODIES Ricardo A. Margni~ 2and Rubkn A. Binaghi ~ Instituto de Estudios de la InmunidadHumoral(CONICET-UBA), Depart~mentode Microbiologia e Inmunologia, Facultad de Farmaciay Bioquimicade la Universidadde BuenosAires. Junin 956, 1113-Buenos Aires, Argentina 2 Centre d’Immunopathologie--AssociationClaude Bernard--, H6pital Saint-Antoine75012Paris, France INTRODUCTION Followingantigenic stimulation, adult vertebrates developa specific cellular and humoral immuneresponse with production of various types of sensitized cells andsoluble antibodiesof different immunoglobulin classes. Theoutstandingcharacteristic of these antibodiesis their ability to combine with a specific antigen. As a consequence of this combination,aggregation of the antibody takes place, and this leads to the triggering of effector immune mechanisms.Giving adequateconditions, the large complexes formedby antibodies and the plurivalent antigens becomeinsoluble andprecipitate in vitro, a reaction on whichthe quantitative determination of soluble antibodies is based. However,it has been knownfor morethan half a century that in most immune sera a populationof antibodies exists that is unable to form precipitates with the antigen, although they can "coprecipitate" in the presence of precipitating antibodies of the same specificity. Anumberof studies madein recent years in our laboratories haveshown that these nonprecipitating antibodies possess an asymmetricstructure, due to a carbohydrategroup present in only one of the two Fab regions of the molecule.Thecombinationof the correspondingantibody site with the antigen is sterically hindered by the carbohydrate group, and as a consequence,the moleculeis functionally univalent. The present review summarizesthe physicochemical and biological 535 0732~)582/88/0410-0535502.00

Annual Reviews

536

MARGNI & BINAGHI

properties of such asymmetric,coprecipitating, or nonprecipitatingantibodies.

Annu. Rev. Immunol. 1988.6:535-554. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

PREPARATION ANTIBODIES

OF NONPRECIPITATING

In 1935 Heidelberger & Kendall (1, 2) madea detailed study of the precipitin reaction. The mainpurposeof their study was to understand, in physicochemicalterms, the modeof combinationbetweenantibody and antigen, whichwasexpected to follow the van der Waalsequilibria laws as well as the Proust and Dalton combinationrules concerningthe proportions of the participants to a chemicalreaction. Theyestablished the experimental conditions giving a precise quantitation of antibody and introduced the notion of equivalence point (zone) where the maximum amountof antibody wasprecipitat6d by antigen. Theyobservedthat the mannerin whichthe antigen was added to the antibody solution greatly influencedthe final result: If the antigen wasaddedin small, successive fractions, the total precipitated antibodywasless than that precipitated whenthe samequantity of antigen wasadded all at once. This indicated that someantibody remainedin solution and was unable to precipitate. Theysubsequently developeda procedureto recover this nonprecipitable antibody and were able to obtain two populations of antibody, precipitating and nonprecipitating (or coprecipitating). In principle, the methodconsists of makingserial additions of small quantities of antigen (1/15 to 1/20 of that correspondingto the equivalencepoint) until no more precipitate is formed. After each addition of antigen the precipitate is separated by centrifugation. Thenonprecipitating antibodyremainsin the final supcrnate and can be quantified by coprecipitation if an antigenantibodyprecipitate of the samespecificity is formedin the solution by addition of newantiserum and antigen. The separation and purification of the nonprecipitating antibodies from the final supernate can nowbe madeeasily by using the techniques of immunoabsorption (3-5). Onthe other hand, the precipitating antibodies can be removedfromthe collected precipitates after eachadditionof antigen. It is then possibleto isolate and purify the precipitating and nonprecipitating antibodies from the same sampleof antiserumin order to study and comparetheir properties. Details of the techniquehavebeen describedelsewhere(3-5). The original work of Heidelberger & Kendall was done with rabbit antibodies. Subsequently,nonprecipitating antibodies were observedin manyother animalspecies, namelyguinea pig (4), rat (6), horse (7), (5, 8, 9), cow(10), donkey(7), human(11), mouseand goat (unpublished

Annual Reviews NONPRECIPITATING ASYMMETRIC ANTIBODIES

537

results). They were found in practically all antisera studied, of many different specificities: hen albumin, serum albumin, gammaglobulin, various haptenated proteins, tetanus toxoid, various bacteria, and parasites, etc.

Annu. Rev. Immunol. 1988.6:535-554. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

PROPERTIES ANTIBODIES

OF THE NONPRECIPITATING

Precipitating and nonprecipitating antibodies were separated by the serial absorption technique and purified by immunoabsorption using affinity chromatography with the insolubilized antigen. In general, the quantity of nonprecipitating antibody obtained represented between 5% and 15% of the total antibody present in the antiserum and was found in all subclasses of IgG in the different animal species studied (3-11). Noinformation is presently available regarding the presence of nonprecipitating antibodies in other immunoglobulinclasses. Precipitating and nonprecipitating antibodies belonging to the same immunoglobulin subclass were always prepared from the same sample of antiserum in order to comparetheir properties.

Physicochemical Properties Various tests were performed to investigate whether precipitating and nonprecipitating antibodies were antigenically different. The results were systematically negative. Antisera were prepared by immunization of rats with either precipitating or nonprecipitating anti-DNP sheep IgG1, and complete identity was found in immunodiffusion tests (12). Furthermore, these antisera could be completely absorbed with either precipitating or nonprecipitating purified antibodies. In immunoelectrophoresis, precipitating and nonprecipitating antibodies of the same specificity isolated from the same antiserum showed identical mobility. Completely identical results were also observed on peptide and diagonal maps(fingerprinting) after enzymaticdegradation, as well as by autoradiography after enzymatic degradation, 2-MEreduction, alkylation with labelled iodoacetamide, and high voltage electrophoresis. Sedimentation coefficients of precipitating and nonprecipitating antibodies were similar--about 7 S--and no differences were found in molecularsize, as determinedby gel filtration (3, 4). The conclusion that can be drawn from all these results is that precipitating and nonprecipitating antibodies have identical structures. Nevertheless, the different behavior of these antibodies needs to be explained, with regard to their ability to precipitate with antigen. The fact that nonprecipitating antibodies coprecipitate and combine

Annual Reviews 538

MARGNI

& BINAGHI

with immunoadsorbents indicates that they have a high affinity for the specific antigen. Nevertheless,they are unableto formthe molecularstructures leading to precipitation. It must be remarked,incidentally, that coprecipitation is a general phenomenon, not restricted to antibodies of the same immunoglobulin class or even the same species, and it always occurs in the presence of antigen-antibodycomplexesof the samespecificity.

Annu. Rev. Immunol. 1988.6:535-554. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Biological Properties The study of numerousbiological functions induced by antibodies has shownthat the complexesformedby nonprecipitating antibody and antigen are unable to activate manysuch functions. This seemsto be a general property whichis certainly related to the inability to form molecular aggregatesof the size required to trigger the immunological mechanisms. Thedetails of these studies are as follows. COMPLEMENT FIXATION Nonprecipitating antibodies are unable to fix complement either by the classical or the alternative pathway.This has beendemonstrated with a variety of purified antibodiesof different classes and from various species (3-5, 11, 13). However,since nonprecipitating antibodies can firmly combinewith antigen, they act in a competitiveway whenthey are mixedwith precipitating antibodies of the samespecificity. Thecomplement-fixing capacity of the mixturesis proportionalto the ratio of precipitating to nonprecipitating antibody. At 20%nonprecipitating antibody, the influence is already significant, and at 80%the complementfixing activity is practically abolished(13-15)(Figure PHAGOCYTOSIS AND OPSONIZATIONIt was found that nonprecipitating antibodies are cytophilic to macrophages in exactly the same wayas precipitating antibodies (11, 13), a result whichsuggests that the Fc regions of both moleculesare functionally similar in this respect. But whenthe "in vitro" phagocyticactivity of the correspondingmacrophages wasinvestigated, it appearedthat only precipitating antibodywasactive. Similarly, purified anti-DNPnonprecipitating antibody injected intravenously in the mousedid not induce the clearance of DNP-labelled circulating Salmonella, while the clearance was immediatewhenprecipitating antibody was injected (11, 13, 16). Again, competition was demonstratedin this system; the value of the phagocyticindex wasdiminished as the ratio of nonprecipitatingto precipitating antibodyincreased. Anexampleis presented in Figure 1. CYTOPHILIA,

ANAPHYLACTIC PHENOMENA Nonprecipitating antibodies do not provoke an Arthusreaction, a result that is in line with their inability to fix comp-

Annual Reviews NONPRECIPITATING

539

ASYMMETRIC ANTIBODIES (B) 4.6

~.00

8o

Annu. Rev. Immunol. 1988.6:535-554. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

60

80/20 40/60 RATIO pp/non-ppAb

I 3

I 6

I 1 9 12 15 TIME (seconds)

Fi#ure 1 Complement fixing activity (A) and blood clearance of antigen precipit~.ting and nonprecipitating antibody mixtures at different ratios.

(B) mediated

lement. On the contrary immediate hypersensitivity phenomenacan be induced by nonprecipitating antibodies. The experiment of Kabat & Benacerraf (17) showedthat precipitating and nonprecipitating rabbit antiovalbuminantibody were equally effective in sensitizing guinea pigs for systemic anaphylactic shock. In passive cutaneous anaphylaxis (PCA), nonprecipitating antibodies appear to be less active than precipitating ones (3). The mechanismof anaphylactic sensitization by IgG antibodies is not completely elucidated. IgG do not bind firmly to the membraneof the basophil or mast cell, and IgG and antigen may first have to form a complexin the fluid phase, which subsequently acts on the cell membrane, inducing degranulation and release of anaphylactic mediators (18, 19). any case, it is evident that nonprecipitating antibodies possess the necessary structures to activated cells but are less efficient in this respect than precipitating antibodies. CVTOTOXIC ACTIVITY The protective activity of precipitating and nonprecipitating antibodies was studied in mice using rabbit anti-Salmonella typhim, urium and cow anti-Brucella abortus (10, 16), Five times more nonprecipitating antibody than precipitating antibody was necessary to obtain 50% protection of the mice challenged with 10 minimumlethal doses of Salmonella. Similarly, the "splenic infection index" was four times higher in mice passively immunized with anti-Brucella abortus non-

Annual Reviews

540

MARGNI & BINAGHI

precipitating antibody than in mice treated with precipitating antibody, 7 days after infection with Brucella 043 vs 43).

Annu. Rev. Immunol. 1988.6:535-554. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

AGGLUTINATION OF SENSITIZED RED CELLS

Nonprecipitating antibodies

were able to agglutinate sheepred cells sensitized with the specific antigen, although less efficiently than precipitating antibodies. Onthe other hand, nonprecipitating antibodies did not agglutinate sensitized humanred cells. WhenF(ab’)2 fragments were tested, the fragments obtained from precipitating antibodies were agglutinating in all cases, whereasthe fragments from nonprecipitating antibodies failed to agglutinate either sheep or humansensitized red cells. These apparently contradictory results became explicable whenit was subsequently found that the red cells of sheep, and manyother animal species, possess a membranereceptor for aggregated Fc (20, 21). Thus, agglutination can be obtained by two different mechanisms, i.e. by attachment of the combiningsites of the antibody molecule to the antigen, or by attachment of the molecule to the Fc receptor present on the red cell. As is discussed later, the nonprecipitating antibody is functionally univalent, but agglutination can be obtained because the antibody can attach to one red cell by a combiningsite and to another red cell via the Fc receptor. Humanred cells do not normally express the Fc receptor, and therefore univalent nonprecipitating antibodies do not agglutinate them. Obviously, no agglutination can be obtained with the F(ab’)2 fragment of the univalent nonprecipitating antibody. The Fc receptor is present in most vertebrates. It is also present in humanred cells but is only expressed after treatment with trypsin. Studies on the characteristics of the Fc red cell receptor, including its properties, purification, phylogeny, and ontogeny, have been published elsewhere (20-

24). The differing behavior of purified nonprecipitating antibody whentested with sheep or humansensitized red cells provides a simple procedure to characterize and detect it, as well as to obtain an approximate estimation of its proportion when dealing with mixtures of precipitating and nonprecipitating antibodies (25). These findings demonstrate that agglutination must be carefully interpreted, especially whenit is performedwith humanred cells, as is frequently the case. Anexampleis presented in Tables 1 and 2 whichreport the results obtained with sera from patients with American trypanosomiasis and cows with brucellosis, analyzed by various serological reactions. The observed discrepancies are due to the presence of nonprecipitating antibodies, detected by IF or ELISAbut not by precipitation or complementfixation. Whenagglutination techniques are used, the results are quite different according to the origin of the red cells used. It is therefore important to

Annual Reviews 541

NONPRECIPITATING ASYMMETRIC ANTIBODIES Table 1 Serological Brucella abortus

analysis

of sera from cows infected

with

Annu. Rev. Immunol. 1988.6:535-554. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Serological reaction Serum

Wright

Coombs

Immunofluorescence

1 2 3 4

1/640 1/20 1/40 1/20

1/1280 1/640 1/5120 1/640

1/1600 1/800 1/3200 1/800

Table 2 Patients infected with Trypanosomacruzi Serological reaction

Serum Precipitation I 2 3 4 5

+++ + _ ---

Complement fixation 1/512 1/64 1/32 1/8 1/4

Elisa 1/800 1/3200 1/400 1/1600 1/800

Immunofluorescence 1/800 1/1600 1/800 1/800 1/800

Haemagglutination HRC SRC 1/640 1/320 1/80 1/80 1/80

1/640 1/640 1/160 1/640 1/320

HRC:sensitized humanred cells; SRC:sensitized sheep red cells.

employa suitable panel of serological tests in order to ensure a correct diagnosis.

PRODUCTION OF NONPRECIPITATING ANTIBODIES As already indicated, the presence of nonprecipitating antibodies has been ascertained in all animal species studied, following immunization with a variety of antigens or after parasitic or microbial infection. In sheep immunizedby repeated intramuscular injections of soluble DNP-BSA for a period of one year, nonprecipitating antibody was producedin all during the immunization and represented about 10%of the total serum antibody (5). This proportion is similar to that generally found in other animal species. Higher concentrations have been observed in rabbits immunized with Salmonella typhimuriumand in cows infected by Brucella abortus (10,

Annual Reviews 542

MARGNI & BINAGHI

16), in which cases nonagglutinating antibodies detected by a Coombs reaction amountedto 25%of the total. This observation suggested that the nature of the antigen could influence the productionof nonprecipitating antibodies. To test this hypothesis rabbits were repeatedly inoculated

Annu. Rev. Immunol. 1988.6:535-554. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

intramuscularly

with soluble ovalbumin or ovalbumin insolubilized

by

treatment with glutaraldehyde or covalently attached to Brucella abortus (26). The results of these immunizationsare presented in Figure 2. It can be seen that the proportion of anti-ovalbumin nonpreeipitating antibody is 25-65%in the case of immunizationwith particulate antigen and only 10-15%whenthe antigen is soluble. Moreover, whenthe nature of the antigen was changedin the course of the immunization,the proportions of nonprecipitating antibody changed concordantly. While the ratio of precipitating to nonprecipitatingantibodyvaried greatly according to the nature of the antigen, the absolute quantity of nonprecipitating antibody wassimilar in all cases. Thesefindings, so far unexplained,indicate that the formof presentation

0.5

~ 4

8

12

16

20

24

Figure 2 Productionof precipitating (O) and nonprecipitating(0) serumantibodies rabbits repeatedlyinoculatedwith soluble henegg albumin(A), egg albumincoupled Brucellaabortus(B) andegg albuminpolymerized by glutaraldehyde (C).

Annual Reviews

NONPRECIPITATING ASYMMETRIC ANTIBODIES 543 of the antigen modulatesthe relative synthesis of precipitating and nonprecipitating antibodies. Theimplications of this fact for defense mechanismsare discussedlater.

Annu. Rev. Immunol. 1988.6:535-554. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

REASONS FOR THE INABILITY PRECIPITATES WITH ANTIGEN

TO FORM

The nonprecipitating antibody moleculebehavesas if it were univalent. Beforegoing into the details of the experimentsdemonstratingthis univalence,it is interesting to recall the varioushypothesesthat wereproposed to explain the nonprecipitability. The most common explanation for many years wasthat nonprecipitatingantibodies representedthe populationwith weakaffinity, unable to form stable bondswith antigen. This hypothesis is contradicted, however,by the fact that nonprecipitatingantibodyfirmly attaches to antigen during coprecipitation and whenimmunoabsorption techniquesare used. Furthermore,the averageaffinity constant, measured with univalent hapten, appearedto be about the samefor precipitating and nonprecipitating antibody from the sameantiserum. Anotherpossibility advancedwasthat nonprecipitating antibody is specific for only one of the manydeterminantsof the antigen molecule,in whichcase, the antigen being univalent, no precipitation could occur (27) This possibility was eliminatedby the use of antihaptenantibodies anda protein carrier highly conjugatedwith the hapten as antigen (3). It mustbe kept in mind,incidentally, that there exist mariynonprecipitatingantibodies, different from those described in this review, whosenonprecipitability is due to the restricted specificity; this is the case for manymonoclonal antibodies. Themost reasonable hypothesis appearedto be univalence of the antibodymolecule. Marrack(28) suggested that nonprecipitating antibodies had a molecularstructure different fromthat of precipitating antibodies, but this suggestion was not supported by physico-chemicalstudies (2931). Other reasons for functional univalence could be a particular distribution of electric chargeson the molecules,inducingelectric repulsion, or the existenceof disulfide bondslimitingthe flexibility of the Fabregions. Still anotherexplanationcouldbe the existenceof an allosteric effect after combinationof one antibody site with one moleculeof antigen, resulting in a diminutionof the affinity of the other site. Wehave previously mentionedthat nonprecipitating antibodies are presentin all subclassesof IgG.Adifferent type of nonprecipitatingantibody has been recently demonstrated in pigs, by Franek & colleagues (32-35); in this case, they belongto a different subclass and differ from precipitating antibodyin manystructural aspects.

Annual Reviews 544

MARGNI & BINAGHI

Annu. Rev. Immunol. 1988.6:535-554. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Equilibriumexperiments,using monovalenthaptenas ligand, haveclarified manyaspects of the combinationof nonprecipitating antibody and antigen. Equilibrium Studies Numerousexperiments have been performed using anti-DNPantibodies. In most cases sheep IgG1 antibodies were used, but the same results wereobtained with antibodies from other species, namelyrabbit and rat. Precipitating andnonprecipitatingantibodies werepurified, as described, from the samepool of antisera, and the IgG1 fraction wasseparated by chromatographyon DEAE-cellulose. F(ab’)2, Fab, and Fab’ fragments were prepared. All these preparations were reacted with the monovalent hapten DNP-eaminocaproicacid and studied by fluorescence quenching and by a radioimmune assay (5, 9, 36). Theresults can be summarized follows: 1. The Scatchard plots obtained with nonprecipitating antibody or its F(ab’)2 fragmentwereclearly bimodal.Thefirst part of the curve, low hapten concentration extrapolated to r = 1, while at high hapten concentration the extrapolation gave a value of r = 2. Whenprecipitating antibodywastested, the curve intercepted the abscissa at a value of 2, in all cases as expected.Examples are presentedin Figure3 and the correspondingKovalues are reported in Table 3. 2. Whenthe Fab fragments obtained from nonprecipitating antibody were applied to a columncontaining immunoabsorbent(polymerized DNPBSA),about 50%were retained (high affinity fraction) and could subsequentlyeluted with the hapten. Theunretained(low affinity) fraction wasstill able to react with the hapten. TheFabfragmentsobtained from predpitating antibody were completely retained by the immunoabsorbent. Theseresults are illustrated in Figure 4, and the values of Koare reported in Table 3. Theinterpretation of these results is that the two Fab regions fromthe samemoleculeof nonprecipitating antibodies are different. Theantibody site present in one of the fab regions has an affinity for the haptenabout 100 times higher than that of the other site. Thedemonstrationthat each moleculehas twodifferent Fb regions comesfromthe fact that the F(ab’)2 is completely retained by the immunoabsorbent,while only 50%of the Fab’ fragmentsobtainedfromit is retained. Themoleculeis, ’therefore, asymmetric. All these experimentswere performedwith a small univalent hapten (DNP).Whennonprecipitating antibody was reacted with a high molecular weight antigen, namelyhighly substituted DNP-BSA, it was found

Annual Reviews NONPRECIPITATING

ASYMMETRIC

545

ANTIBODIES

Radioimmunoassay

Fluorescense quenching F (ab’)

IgGl

F[ab’) 2

Annu. Rev. Immunol. 1988.6:535-554. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

5

1 IgGl 10

2

2 i F [~b’ }2

i0

i F(ab’) 2

2

20

20 16

6

12

12 8

4 4

2

i

2

I

2

I

2

i

2

Figure3 Scatchard plots of the interactionbetweenDNP-EACA andIgG1sheepantiDNP precipitating andnonprecipitating antibodies andtheir F(ab’)2fragments, as measured byfluorescence quenching andbyradioimrnunoassay. thatthe curve extrapolated at r = 1, even at high concentrations of antigen. With precipitating antibody, extrapolation gave a value of r o = 2. This is in contrast to what was found when a univalent hapten was employed and indicated that one molecule of nonprecipitating antibody cannot react with more than one antigen molecule of high molecular weight.

Structural Aspects The above results indicate that the molecule of nonprecipitating antibody is asymmetric, and that the different affinity constants of their antibody sites make it functionally univalent. This adequately explains whythe antibody is unable to form precipitates with the antigen. The chemical and physicochemical properties of precipitating and non-

Annual Reviews

546

MARGNI & BINAGHI

Table 3 Association constant (K~) of sheep anti-DNP precipitating bodies and their F(ab’): and Fab’ fragments.

and nonprecipitating

anti-

Association constant (K0)* Fluorescent quenching

Annu. Rev. Immunol. 1988.6:535-554. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Precipitating

Nonprecipitating

9.5 x 107 -~ M -I 5.0 × 107 M 9.0 × 107 -I M

Whole F(ab’)z Fab’ Whole F(ab’)2 "Wab’ :~Fab’

Radioimmunoassay

Highaffinity 2 x 106 -l M 2.1 x 106 -~ M 3.2 × 106 -~ M

Lowaffinity 1.6 × l0 4 -1 M 1.2 x 104 -l M

6.3 x 107 -~ M -I 3.5 × 107 M 5.0 × 107 -I M Highaffinity 1.2 × 107 -I M 1.2 × 107 -1 M 6.3 x 106 -~ M

Lowaffinity 0.9 × 105 -1 M 1.0 × 105 -l M

-l 2.3 × 104 M

-1 3.2 × 104 M

* Ligand: DNP-EACA. "~Fab’ fragment retained by immunoabsorption. :~Fab’ fragment not retained by immunoabsorption.

precipitatingantibodiesappearto be identical; moreover, the accumulated data on aminoacid sequences of H and L chains, as well as genetic considerations,uniformlysupportthe existence of a symmetric molecule. Onewayto explain the asymmetry wouldbe that nonpeptideparts of the molecule,such as carbohydrate, couldcreate a steric hindrancein one of ~

~ (B)

(A)

(c)

6

4

2

0,5

r

I 0.5

r

I

Figure 4 Scatchard plots of the interactin between DNP-EACA and sheep anti-DNP Fab’ fragments from precipitating antibody (A) and from nonprecipitating antibody (B) affinity sites; (C) low affinity sites), as measuredby fluorescence quenching.

Annual Reviews

Annu. Rev. Immunol. 1988.6:535-554. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

NONPRECIPITATINGASYMMETRIC ANTIBODIES

547

the combiningsites. Experimentswere madeto test this hypothesis, and the results confirmedthat the asymmetry of the nonprecipitatingantibody moleculeis due to a steric hindrance(37). As it was shownbefore, nonprecipitating antibody is unable to precipitate with highly substituted DNP-BSA. However,precipitationoccurred if the DNPdeterminant was separated from the carrier by a short aliphatic chain. This wasdone by using a spacer, ~-aminobutyric acid (GABA).Whenthe purified nonprecipitating antibody was reacted with DNP-GABA-BSA, a precipitate wasobtainedand complement wasfixed. It can be deduced,therefore, that the DNP groupscoupledto the lysine residues of the protein are unableto react with the antibodysite with lowaffinity becausethe antigen molecule cannot get close enough. On the other hand, whenthe DNPgroups are farther apart, as in the case of DNP-GABA-BSA, the access to the combiningsite maybe possible. Nevertheless, the Scatchardplot obtained with nonprecipitating antibody and DNP-GABA-BSA is bimodal, with the two slopes extrapolating to r = 1 and r = 2, indicating different affinities of the antibodysites. To explorethe possibility that a carbohydratemoietycould be producing the steric hindrance, the nonprecipitating antibodymoleculewastreated with an enzyme, endo-fl-N-acetylglucosaminidase H, which splits the carbohydrategroupsattached to the Asnresidue of the peptide chain (37). The enzymatic treatment modified the molecule in such a waythat it becomeprecipiting. The same result was found after treatment of the F(ab’)2 fragment. Also, after enzymatictreatment, the nonprecipitating antibody was able to fix complementwith DNP-BSA as antigen. The Scatchard plots correspondingto these experimentsare shownin Figure 5. Chemicalanalysis of the nonprecipitating antibody indicated that it contains morecarbohydrateresidues than the precipitating antibody, it suggested the presence of an extra residue with a high anannosecontent (R. Binaghiand colleagues, unpublishedresults). This was confirmed affinity chromatographywith concanavalin A. WhenF(ab’)2 from the nonprecipitating antibody was applied to a columnof ConA-Sepharose, it wascompletelyretained, while F(ab’)2 fromprecipitating antibody was not. WhenFab’ was used, the ConA-Sepharoseretained only 50%of the fragment from nonprecipitating antibody. The carbohydrate residue is present in the Fd region of the H chain of the molecule(38). In effect, after reduction andalkylation of nonprecipitatingand precipitating Fab’ fragments, only the Fd fragmentobtained fromthe low affinity Fab’ was retained by ConA-Sepharoseand inhibited the fixation of concanavalin Ato guinea pig red blood cells. Theseresults wereobtained using antibodies from sheep and rabbit.

Annual Reviews 548

MARGNI

& BINAGHI

Annu. Rev. Immunol. 1988.6:535-554. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

(a)

(b) F(ab’)2 fragment from IgGl

i

2

Figure5 Interaction betweenDNP-GABA and sheepanti-DNPantibodyand its F(ab’)2 fragment.Dataobtainedby fluorescencequenching.Precipitating antibody(0); precipitatingantibodytreated withendoglycosidase (O); nonprecipitating antibody(I); precipitatingantibodytreatedwithendoglycosidase (V]).

It can be concluded, therefore, that the asymmetryof the molecule of nonprecipitating antibody is due to a carbohydrate moiety present in only one of the Fab regions. This carbohydrate affects the reaction betweenthe combiningsite and antigen and renders the antibody molecule functionally univalent. The existence of an incomplete glycosylation in immunoglobulins has already been described in other cases. Hinrichs & Smyth (39) in rabbit IgG, Matusuuchi et al (40) in a dextran-binding mouse plasmocytoma, Savvidou et al (41) in humanmonoclonal IgG1, Weitzman& Scharff (42) in a mutant of the mouse MPC-11,and Green & Gleiber (43) in kappa chains. Various explanations have been suggested to account for these observations, including limitations on the availability of the enzymes involved in glycosylation, or competition between folding of the nascent polypeptide and attachment of carbohydrate.

PHYSIOLOGICAL ROLE OF NONPRECIPITATING ANTIBODIES The existence of a significant quantity of asymmetric, nonprecipitating antibodies in most antisera of all animal species studied suggests that they

Annual Reviews

Annu. Rev. Immunol. 1988.6:535-554. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

NONPRECIPITATING ASYMMETRIC ANTIBODIES

549

play someimportant physiological role. For the momentno evidence is available concerningthe nature of this role, but there exist a numberof observations madein recent years that can be interpreted as indicating somefunction of nonprecipitatingantibodies. It is interesting to review these observationsin order to suggestsomelines for future research. Theessential characteristic of nonprecipitatingantibodiesis their incapacity to activate effector mechanisms, andin this sensethey act as inactive antibodies. Whateveris the mechanism by whichthe activation is achieved, it is clear that the lack of efficacy is a direct consequence of the inability to form stable antigen-antibody aggregates of the necessary molecular structure. Nevertheless,nonprecipitatingantibodies bind to antigen with the sameaffinity as do precipitating antibodies and competewith them, so that nonprecipitatingantibodies do not act as indifferent partners in the triggering of immunereactions but rather as blocking antibodies. This has been clearly demonstratedin the case of complementfixation, opsonization,cytotoxicity, etc. In all cases, whatappearsto be important is not the absolute quantity of nonprecipitatingantibodybut the relative proportion of nonprecipitating to precipitating antibody, acting simultaneously. The blocking antibodies maybe beneficial or harmful to the host, accordingto the nature of the antigenand the particular situation in which they act. Blockingantibodieshavebeendemonstratedin allergic conditions and are currently implicatedin the clinical improvement followingspecific antiallergen therapy. Recent workhas indeed shownthat IgG4antibodies producedby allergic patients behave as univalent, thus blocking the antigen. It will be interesting to knowwhetherthe univalenceis dueto a molecular asymmetry. Blockingantibodies have beenfound in various microbial and parasitic infections (14, 15, 44-48). Thereal significance of these antibodies unclear: Theycould act to favor escape mechanisms of the invader or on the contrary could help to prevent anaphylactic reactions in individuals wheresensitized cells (basophils, mastcells) and parasitic antigens are present simultaneouslyin the blood. Still another case in whichblocking antibodies havebeen clearly demonstrated is in tumors(49). Antitumorantibodiescan enhanceor facilitate tumorgrowthand have beenconsequentlydesignated "facilitating" antibodies(50). In these examples,antibodies combinewith antigen but do not activate phagocytic or cytotoxic reactions. At the sametime they prevent, by a competitiveeffect, the action of other phagocyticor cytotoxicantibodies. It has beenshown,in someinstances, that these blockingantibodiesbelong to an immunoglobulin subclass that does not fix complement and does not

Annual Reviews

Annu. Rev. Immunol. 1988.6:535-554. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

550

MARGNI & BINAGHI

trigger a cytotoxic reaction. Thepossibility cannotbe excluded,however, that other characteristics of these antibodies are relevant to the blocking phenomenon. Thereis anotherphysiologicalsituation, so far little exploredfromthe immunological point of view, whereblocking antibodies certainly havea predominantrole: pregnancy. It is well knownthat the pregnant mother producesantibodies against the father’s antigens, and it has been demonstrated that these antibodies fix to the placenta by combiningwith the antigenspresentin it. In summary,it can be said that manydifferent phenomena exist where antibodiesact in a paradoxicalway(i.e. they fail to inducethe elimination of the foreign, specific antigen andinstead protect it. This protection may be helpful (allergies, pregnancy)or harmful(tumors, infections) to host. Muchmoreresearch on the nature of these antibodies is necessary to knowwhether someof these properties can be explained on the basis of an asymmetricalstructure. Anotherinteresting problemis the regulation of the synthesis of asymmetric nonprecipitating antibodies. Wehavementionedthat they generally constitute about 10%of the total antibodypopulationbut their proportion can be muchhigher in special situations, for instance after prolonged immunization with particulate antigens. It is noteworthythat this is precisely the situation prevailing in chronic infections, tumors, pregnancy, etc, whereblocking antibodies have been found. Also, the observations madein mice treated with mixtures of precipitating and nonprecipitating anti-Salmonellaand anti-Brucella antibodies indicate that the cytotoxic effect of these mixturesis inversely proportional to their relative concentration of nonprecipitating antibody. Theantisera employedin these experimentswere producedby infected animals and contained significant amountsof nonprecipitatingantibodies. It is conceivablethat the defense mechanismsin these animals were hamperedby the presence of the nonprecipitating antibody.Thesamecan be said of the humanpatients infected by Tripanosoma cruzi. Lastly, it is temptingto speculate about the role that nonprecipitating asymmetricantibodies mayhave in the regulation of the immunesystem itself, includinginductionof tolerance, stimulationor depressionof immunocompetent cells, modulationof the idiotypic network,etc. CONCLUSIONS The recent studies of nonprecipitating antibodies have provided much informationconcerningtheir properties. This can be ’summarizedas follows:

Annual Reviews

Annu. Rev. Immunol. 1988.6:535-554. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

NONPRECIPITATING ASYMMETRIC ANTIBODIES

551

--Nonprecipitating IgG antibodies are present in practically all mammalianantisera and in all IgGsubclasses. --Nonprecipitatingantibodies are unableto precipitate with specific antigen, but they firmly attach to it and coprecipitate in the presenceof precipitating antibodyof the samespecificity. Theisolation and purification of nonprecipitating antibodies can be easily done by immunoabsorption techniques. --Nonprecipitating antibodies do not fix complement by the classical or the alternative pathways,they are not cytotoxic; and they do not induce phagocytosisor opsonization, although they are cytophilic to macrophages. However,since they combinewith the antigen, they act in a competitive waywhenmixedwith precipitating antibodies of the same specificity. --Nonprecipitating antibodies do not provokeArthus reactions, but they can induce immediatehypersensitivity phenomena,although they are probablyless active than precipitating antibodies. --Nonprecipitatingantibodies agglutinate sensitized erythrocytes of most vertebrates. Agglutination is produced by attachment of non-. precipitating antibody to the sensitized cell not only by the specific antibody site but also via an Fc receptor present on the red cell membrane.Human red cells do not havethis receptor, and consequently nonprecipitating antibodies do not agglutinate them. --Nonprecipitating antibodies represent about 10%of the total serum antibody, but their proportion can be greatly increased by repeated immunizationwith particulate antigens. --Equilibrium studies of the combination between antigen and nonprecipitating antibodies indicate that the latter have, in the same molecule, one antibody site of high affinity and another site of low affinity. Thedifference in their associationconstantsis about100fold. In consequence,the moleculeacts as functionally univalent. This fact explains the nonprecipitabilityand lack of activation of immune effector mechanisms. --The low affinity of one antibody site is due to the presence, in the correspondingFab region of the molecule,of a prosthetic carbohydrate group whichsterically hinders on the combinationwith antigen. The moleculeis, then, asymmetric. --The nonprecipitating or coprecipitating or univalent or asymmetric antibodies play a role as blocking antibodies in a numberof physiological and pathological situations. Theymaybe operative in allergy, parasitic, and microbial chronic infections, tumorimmunity,and pregnancy. They mayalso have a role in the regulation of the immune response.

Annual Reviews

552

MARGNI& BINAGHI

Manyquestions are prompted by these results, which can only be answeredby careful and detailed research. Themostobviousneedis for determiningthe precise structure of the asymmetricmoleculeand its modeof synthesis, and the physiological and pathological role of the nonprecipitatingantibodies.

Annu. Rev. Immunol. 1988.6:535-554. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

ACKNOWLEDGMENT

This workwas supported in part by a scientific cooperation agreement between CONICET (Argentina) and INSERM (France). Literature Cited 1. Heidelberger, M., Kendall, F. E. 1935. J., Hajos,S. E., Veira, S., Manghi,M., A quantitative theory of the precipitin Bazzurro, M. 1977. Non-precipitating reaction. III. Thereactionbetweencrysantibodies isolated by immunotalline egg albuminand its homologous adsorption. Immunochemistry14:299 antibody. J. Exp. Med.62:697 10. Parma, A. E., Santisteba, G., Margni, 2. Heidelberger, M., Treffers, H. P., R. A. 1984. Analysisand in vivo assays Mayer,M. 1940. A quantiative theory of cattle agglutinating and non-aggluof the precipitin reaction. VII. Theegg tinating antibodies.Vet. Microbiol.9:391 albumin-antibodyreaction in sera from 11. Perdig6n, G., Margni,R. A., Gentile, the rabbit and horse. J. Exp. Med.71: T., Abat~ngelo, C. 1982. Humananti271 tetanus toxin precipitating and copre3. Margni, R. A., Binaghi, R. A. 1972. cipitating antibodies. Immunology45: Purification and properties of non-pre183 cipitating rabbit antibodies. Immu- 12. Ronco,J., Sciutto, E., Leoni,J., Margni, nology 22:557 R. A. 1984. Structural studies of sheep 4. Margni,R. A., Hajos, S. E. 1973. BioIgG1precipitating and coprecipitating logical and physicochemicalproperties antibodies. Vet. ImmunoL Immuof purified anti-DNPguinea pig nonnopathoL 5:369 precipitating antibodies. Immunology 13. Margni, R. A., Perdig6n, G., Abafin24:435 gelo, C., Gentile, T., Binaghi, R. A. 5. Margni,R. A., Paz, C. B., Cordal, M. 1980. Immunobiologicalbehaviour of E. 1976. Immunochemical behaviour of rabbit precipitating and non-presheep non-precipitatingantibodies isocipitating (coprecipitating) antibodies. lated by immunoadsorption. ImmuImmunology41:681 nochemistry13:209 14. Carbonetto,C. H., Hajos, S. E., Margni, R. A., Esteva, M., Cristopoulus, C. 6. Crespo,O., Margni,R. A. 1978.An~lisis 1983.Estudiosserol6gicos en pacientes de la capacidad reactiva y grado de con enfermedad de Chagas cr6nica. alteraci6n de los anticuerpos copreMedicina43:131 cipitantes anti-DNPocurrida durante la purificaci6n. Acta Bioquim.Clin. Lati- 15. Carbonetto, C. H., Malchiodi, E., Margni,R. A. 1986. IgG antibody type noamer.12:435 in sera from patients with chronic 7. Cordal, M.E., Margni,R. A. 1974.IsoChagas’ disease. Immunologia(Barlation, purification andbiological propcelona) 5:18 erties of horse precipitating and nonprecipitating antibodies. Immunochestry 16. Margni, R. A., Parma, A. E., Cerone, S., Erpelding, A., Perdig6n, G. 1983. 11:765 Agglutinating and non-agglutinating 8. Margni,R. A., Castrelos, O. D., Paz, C. antibodies in rabbits inoculated with a B. 1973. The sheep immuneresponse. particulate antigen (SalmonellatyphiVariationofanti-haptenand anti-cartier murium). Immunology48:351 antibodies in the ~,~ and ~’2 immunoglobulin fractions. Immunology24: 17. Kabat,E. A., Benaeerraf,B. 1949.Passive anaphylaxisin the guinea pig. IV. 481 9. Margni, R. A., Cordal, M. E., Leoni, Passive sensitization with non-pre-

Annual Reviews

Annu. Rev. Immunol. 1988.6:535-554. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

NONPRECIPITATINGASYMMETRIC ANTIBODIES

553

cipitable or "univalent" rabbit antiF. 1964. Valenceand affinity of equine ovoalbumin.J. Immunol.62.97 non-precipitatingantibodyto a haptenic group. Science 146:401 18. Binaghi, R. A., Liacopoulos, P., Halpern, B. N., Liacopoulos-Briot, M. 30. Klinman, N. R., Karush, F. 1967. Equine anti-hapten antibody. V: The 1962. Interference of gammaglobulins non-precipitabilityof bivalent antibody. with passive in vitro anaphylacticsenImmunochemistry4:387 sitization of isolated guinea-pigintes31. Carter, B. G., Harris, T. N. 1967. Nontine. Immunology5:204 precipitating rabbit antibodyto hapten: 19. Binaghi,R. A. 1968.Thesensitization of purification andproperties. Immunology tissues andinterference of non-specific gamma globulin. In Biochemistryof the 12:75 Acute Allergic Reactions, ed. K. F. 32. Fran~k,F., Doskocil,J., Simek,L. 1974. Austen, E. L. Becker, p. 53. Oxford: Different types of precipitating antibodies in early and late porcine antiBlackwell’sSci. 20. Margni,R. A., Hajos, S. E., Manghi,M. dinitrophenyl sera. lmmunochemistry 11: A., Perdigrn, G., Leoni, J. 1974. Red 803 cell Fcreceptorsand their participation 33. Fust, G., Medgyesi,G. A., Franrk, F. in the passive haemagglutination 1977. Complement consumption by mediated by non-precipitating antiimmunecomplexes containing various bodies. Immunology27:863 pig anti-DNP antibodies and DNP21. Hajos, S. E., Margni,R. A., Perdigrn, serum albumin. Immunochemistry14: G., Manghi,M., Olivera, R. 1978. Bind259 ing of immunoglobulins and immune 34. Fran~k, F., Olsrvsk~, Z., ~imek, L. complexes to erythrocytes of 1979. Non-precipitatingpig anti-dinitrophenyl antibody:factors influencing vertebrates, lmmunochemistry 15:623 22. Margni,R. A., Hajos, S. E., Perdigrn, the conversioninto precipitating antiG., Manghi,M., MalanBorel, I. 1979. body. Eur. J. Immunol.9:696 Ontogenicevolution of chickenred cell 35. Loseva,O. I., Tischenko,V. M., OlsbvFc receptor. Cell. Immunol.48:235 sk/t, Z., Franek, F., Zav’yalov,V. P. 1986. Correlation of the character of 23. Man.ghi,M., Venturiello, S. M., Etcheverngaray, M., Margni, R. A. 1987. intramolecularmeltingwithdigestibility Isolation andpartial characterizationof by pepsin in precipitating and non-prebiological active Fc receptor of chicken cipitating pig anti-Dripantibodies.Mol. red cells. Biochem.Biophys. Acta 923: Immunol. 23:743 381 36. Ronco,J., Sciutto,E., Leoni,J., Margni, 24. Manghi,M., Gutierrez, M. I., EtchR. A., Binaghi,R. A. 1984. Interaction everrigaray, M., Margni, R. A. 1987. of purified precipitating and non-prePreparation of rabbit anti-chicken red cipitating (coprecipitating) antibodies cell Fc receptor serum. Analysisof the with hapten and with haptenated cell membranereceptor distribution. protein. Evidence of an asymmetrical Inmunologia(Barcelona) 6:64 antibody molecule. Immunology52:449 25. Margni,R. A., Hajos, S. E., Cordal,M. 37. Labeta,M.O., Margni,R. A., Leoni,J., E., Quiroga, S. 1976. The haemagBinaghi, R. A. 1986. Structure of asymglutinating activity of different antimetric non-precipitatingantibody:presDNPantibody populations when dinience of a carbohydrateresidue in only trophenylatedsheep and humanred cells one Fab region of the molecule. Immuare used as agglutinogen. J. Immunol. nology 57:311 Methods13:51 38. Leoni, J., Labeta, M., Margni,R. A. 26. Margni,R. A., Perdigrn, G., Gentile, 1986. The asymmetric IgG non-preT., Abat~ingelo,C., Dokmetjian, J. 1986. cipitating antibody.Localizationof the IgG precipitating and coprecipitating oligosaccharide involved in that antibodiesin rabbits repeatedlyinjected phenomenonby concanavalin A interwith soluble and particulate antigens. action. Mol. Immunol.23:1397 Vet. Immunol.Immunopathol.13.51 39. Hinrichs, W. A., Smith, D. G. 1970. 27. Christian, C. L. 9170.Characterof nonStudies on the asymmetricallyattached precipitating antibodies. Immunology oligosaccharide of rabbit immu18:457 noglobulinG. Biosynthesisandstability 28. Marraek,J. R. 1961. In Immunological of the C2-oligosaccharide. Immunology Approaches To Problems in Micro18:759 biology, ed. M. Heidelberger, O.J. 40. Matsuuchi,L., Wims,L., Morrison,S. Plescia. NewBrunswick, NJ: Rutgers L. 1981.A variant of the dextran-bindUniv. Press ing mouseplasmocytoma J 558 with alt29. Klinman,N. R., Rockey,J. H., Karush, ered glycosilationof its heavychain and

Annual Reviews

Annu. Rev. Immunol. 1988.6:535-554. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

554

MARGNI& BINAGHI

R. A., Esteva,M., Segura,E. 1982.Puridecrease reactivity with polymeric dextran. Biochemistry20:4827 fication and biological properties of 4I. Savvidou, G., Klein, M., Horne, C., anti-Tripanosomacruzi antibodies isolated from patients with Chronic Hofmann,T., Dorrington, K. J. 1981, Chagas’disease. Immunol.Lett. 4:199 A monoclonal immunoglobulin G1 in which some molecoles possess gly47. Ball, P. A. J., Bartlett, A, 1969.Serocosilated light chains--I. Site of glylogical reactions to infections with Neccosylation Mol. Immunol.18:793 ator americanus.Trans. R. Soc. Trop. 42. Weitzman, S., Scharff, M. D. 1976. Med. Hyg. 63:362 Mousemyelomamutants blocked in the 48. Bennex,J., Ghilhon, J., Barnabe, R. assembly,glycosilation and secretion 1973. Etude comparative des diverses and immunoglobulin. J. Mol. Biol. 102: methodes de diagnostic immuno237 serologique de la Fasciolose hepatobiliaire experimentale du muton et 43. Green, M., Gleiber, W. E. 1980. Coinfluencedu traitment sur la persistence translational cleavageandglycosylation of the mineral oil-induced plasdes anticorps. Bull. Soc. Pathol. Exot. 66:116 mocytoma-46BK chain precursor by plasmocytoma microsomes, Arch. 49. Hajos,S. E., Alvarez,E., Pier~.ngeli,S., Biochem.Biophys. 199:47 Pasqualini, C. D., Margni,R. A. 1984. Antibodies against a tumor associated 44. Forget, A., Borduas, A. G. 1977. An immunobiological enhancement pheantigen in an AKRlymphoma connomenon in experimentalbrucella infecditioned to growin a BALB/c mice. Vet. tion of the53:190 chick.Int. Archs.AllergyAppl. Immunol. Immunopathol.7:53 Immunol. 50. Voisin, G. A. 1983. Immunological intervention of the placentain maternal 45. McCutchan,D., Katzenstein, D., Norimmunological tolerance to the foetus. quist, D., Chikami,G., Wunderlich,A., In ReproductionImmunolotTy,ed. S. I. Braude, A. I. 1978. Role of blocking antibody in disseminated gonococcal Sojima, W. D. Billington, p. 121. infection. J. lmmunol.121:1884 Amsterdam: Elsevier Sci. 46. Hajos,S. E., Carbonetto,C. H,, Margni,

Annu. Rev. Immunol. 1988.6:535-554. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Annu. Rev. Immunol. 1988.6:535-554. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Annual Reviews www.annualreviews.org/aronline

Annu. Rev. Immunol. 1988.6:555-580. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Ann. Rev. Iramunol.1988.6: 555~80 Copyright©1988by AnnualReviewsInc. All rights reserved

THREE-DIMENSIONAL STRUCTURE OF ANTIBODIES P. M. Alzari, M.-B. Lascombeand R. J. Poljak Drpartement d’Immunologie, Institut 75724 Paris Cedex 15, France

Pasteur,

INTRODUCTION The general principles of the genetic control and the three-dimensional structure of antibody molecules have been workedout in the last 20 years. This very exciting period has seen the unraveling of the mechanismsthat generate diversity in a molecule which participates in an adaptive response to varied environmental conditions. This diversity is superimposed on a pattern of genetic transmission of constant hereditary traits and, in molecular terms, a constant molecular structure. The antibody molecule has thus been repeatedly presented as a paradigmof biological flexibility; its functional and structural individuality is superimposed on a carefully conserved structure. However, not all the problems posed by antibody function and specificity have been worked out. In fact, the search for a detailed understanding of the structural bases of specificity and the physicochemicalcharacterization of antibody action at the molecular level are only beginning. Waysof approaching these problems have been cleared by recent developments in the characterization of immuneprocesses and of the cells and molecules that are the effectors of those processes. In addition to that research, there is a continuing search for a molecular definition of immunogenicityand antigenicity, for new uses of antibody molecules in regulating immuneresponses, in mimickingforeign antigens, and in delivering therapeutic agents, etc. These "technical" aspects of antibody action have in the last few years attracted muchattention. Thus, the possibility of producing vaccines with artificial antigens has been an inducementto the study of antigenicity, to the determination of the mini555 0732-0582/88/0410-0555502.00

Annual Reviews ALZARI,

LASCOMBE & POLJAK

mummolecular bases of antigenicity, bodies.

and to antigen recognition by anti-

Annu. Rev. Immunol. 1988.6:555-580. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

OVERALL THREE-DIMENSIONAL OF ANTIBODIES

STRUCTURE

X-ray diffraction studies of crystalline antibody fragments, light (L) chains, and myelomaimmunoglobulin (Ig) molecules form the bases our knowledgeof the detailed three-dimensional structure of antibc~dies. Thesestudies have been reviewed before in this series (1, 2). Since the above mentioned reviews, the three-dimensional structure of another humanIg, Mcg(IgG1, 2), has been determined by X-ray diffraction techniques to 6.5 ~ resolution (3). In this IgG, as in the previously reported IgGDob(4) a deletion of the hinge region brings the Fab and regions closer together. The L (2) chains are disulfide bonded to each other, since the half cysteines of the heavy (H) chain to which they are normally bonded are deleted. The molecule has an exact two-fold axis of symmetry, which can be described as T-shaped although the angle between the Fab arms in 170° instead of the 180° value observed in IgG Dob (4). Evidently, the absence of a hinge region restricts the relative intersegmental mobility of Fab and Fc. This mobility had been observed before even in the crystalline state in human IgGl Kol (5). A second example intersegmental mobility can be added, that of IgG2(~) Zie, a cryoglobulin with four inter-H chain disulfide bonds in its hinge region. This IgG2 and its F(ab’)2 crystallize isomorphically and give similar X-ray intensity distributions, indicating that the Fc assumesmultiple orientations as if in motion relative to the Fab arms of the molecule (6). Crystals of the humanH-chain disease protein Riv have given further evidence for conformational flexibility in the hinge region of IgG (7). Riv is a yl protein with a deletion of entire VHand CH1 domains. Its homogeneousN-terminal sequence starts at position 221; it includes most of the hinge region missing in Fc and extends to the C-terminus of CH3. However,its crystals are i~somorphouswith those of humanFc, indicating that the hinge region and the initial part of the CH2 domaindo not assume a unique conformationin the crystalline state. The gross conformation of humanIg belonging to the subclasses IgGl, IgG2, IgG3, and IgG4 has been studied by electron microscopy (8) and, in solution, by sedimentation and small angle X-ray scattering (9). The latter studies could be briefly summarizedby saying that they indicate a correlation between the lengths of the hinge region sequences and the structure of the different classes of Ig. The proposedoverall structures are

Annual Reviews

Annu. Rev. Immunol. 1988.6:555-580. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

STRUCTURE OF ANTIBODIES

557

(a) extended with noncoplanar Fab arms for IgG1, (b) more compact noncoplanar arms for IgG2, (c) extended with a hinge region of about 90/~ for IgG3, and (d) T-shaped with little or no hinge region for IgG4. These conformations can be correlated in very general terms with the activity in effector functions displayed by antibodies belonging to the different subclasses. Thus, IgG2 and IgG4 have short hinge segments and are poor mediators of effector functions such as the activation of complement,contrary to the long-hinged, more intersegmentally flexible IgG1 and IgG3. Mouse monoclonal antibodies (mAbs) also exhibit good correlation betweensegmental flexibility and their ability to activate complement: IgG1 < IgG2a < IgG2b (10). Immuno-electron microscopy studies (11, 12) have shown a rotational flexibility around the long axis of Fab. This flexibility of antibodies may be important for their roles in effector functions and in facilitating the binding of equivalent antigenic determinants by the same antibody molecule when they are randomly oriented in space. A model for the IgGE Fc region has been proposed (13), based on the assumption that the domainsof the structure should be closely related to the constant domains of the vl Fc whose three-dimensional structure is known(14). In this model of IgGE, CE2and CE4are laterally paired CH3in IgG1, and the Cn3 is arranged as the CH2domains of IgG1. In the absence of an experimentally determined structure, this model provides a tentative three-dimensional frame of reference to discuss properties of IgE. THE

IMMUNOGLOBULIN

SUPERFAMILY

The Ig-fold (15, 16) established by X-ray crystallography has been analyzed and redescribed in different publications emphasizing Ig chain dimerization (17), the structure of the presumedantigen combiningsite (18), and other structural features of antibodies. However,its most interesting aspect from the point of view of evolutionary biology is that the Ig-fold is more ubiquitous than first thought. The Ig-fold occurs in a number of molecules most of which have no direct role in immuneprocesses and indeed which belong to cells of other systems such as neurones. Although the folding scheme was found in Igs, sequence homologyshowed that it also occurs in other molecules directly or indirectly involved in antigen recognition: the ~, fl, and V chains of T-cell antigen receptors; the heterodimers of class I and II of the major histocompatibility complex (MHC), and the T-cell molecules CD2, CD3, CD4, and CD8(reviewed Ref. 19). The three-dimensional structure of fl2-microglobulin, a chain associated with class I MHCmolecules, has been determined by X-ray crystallography (20). As expected, its folding correspondsto that of a free-

Annual Reviews 558

ALZARI,

LASCOMBE & POLJAK

Annu. Rev. Immunol. 1988.6:555-580. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Ig domain. The numberof proteins displaying sequence homologywith Igs but whichdo not participate in antigen recognition is increasing. Among these wehave the poly-Ig receptor moleculeactive in transporting Igs across mucosalmembranes (21), the Thy-1(22) and OX-2(23) antigens of unknownfunction present in lymphocytesand in neurones, the glycoprotein of humanplasma (24) whosefunction is unknown,and the N-CAM or neural cell adhesion molecule (25). Anup-to-date treatment of this subject is foundin the chapter on the Ig superfamilyin this volume (26). HIGH RESOLUTION AND L CHAINS

STRUCTURES

OF FABS

Thestructures of someadditional Fabs have been reported. That of the galactan-binding Fab J539 from a murineigA-x has been determined and refined (27) at 2.6 Aresolution. In this crystal structure, the anglebetween the constant and variable parts of Fab or "elbow bend" is 145°. The antigen-combiningsite of J539 is in close spatial proximity to the CH1 constantregion of a neighboringmoleculein the crystal lattice, explaining whyattemptsat diffusing galactan into the crystals wereunsuccessful.The complementarity-determiningregions (CDR)of J539 delineate a large pocket. VL residues Trp90and Tyr92 and VHresidues Trp33 and His52 line sides of the pocket. This conformationsupports the conclusionsfrom a binding study (28, 29) of a numberof deoxy-fluorogalactosides implicating two solvent-exposedTrp residues in the interactions of the J539 combining-site with galactan. Several tentative structural modelsfor such binding could be constructed using different conformationsof galactan and fitting themto the binding site of J539 to obtain reasonablevan der Waalscontacts. Oneof these modelsis in general terms similar to that proposedby Glaudemans and colleagues (28, 29). With the determination of the structure of Fab J539, at least four Fab structures knownto high resolution have nowbeen well refined (1, 2). The others are Fab Kol (humanIgG1, x), Fab McPC603 (murine IgA, x) recently refined (30) 2.7/~ resolution, and Fab New(humanIgG1, 2). Other Fabs that have beenstudied or are understudy in different laboratories should achieve a similar degreeof precision in atomiccoordinatesin the near future. Thelight (L) chain dimerLocpresents an interesting case of quaternary structural variation on an otherwiseconstanttertiary structure. Thethreedimensionalstructure of this L (2) chain dimer has beensolved by X-ray crystallographic techniques to 2.0 ~ resolution (31). The two Vudomains are related by a pseudotwo-foldaxis, but they havea translational corn-

Annual Reviews

Annu. Rev. Immunol. 1988.6:555-580. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

STRUCTURE OF ANTmODIES

559

ponent (3.5/~) along that axis which is clearly longer than that observed in the Fab fragments discussed above where that value is of about 1 ~ or less. The authors describe the resulting "binding pocket" as a protruding site and propose this different type of association as one that could increase the diversity of antibodies and could also occur in the T-cell antigen receptor. Crystals of this light chain dimer grown from a different solvent-distilled water instead of 1.5 Mammonium sulfate--were recently studied to 3.5/~ resolution (32). In this crystal form the VL-VL pairing is similar to those of the VL dimer Mcgand the VH-V L of Fabs; in addition, the combiningsite resembles a more "conventional" antigen binding site. Several Fab structures are under investigation in different laboratories. Fab HED10, from an IgG2a monoclonal antibody (33) and Fab BY04-01 (34) are interesting because they bind single stranded DNAfragments. The three-dimensional structure of an antineuraminidase Fab from the mAbSl0/l has been determined to 3 .~ resolution (35). S10/1 binds neuraminidase from influenza virus strains isolated between 1961 and 1972. The binding patterns indicate that the epitope recognized by S10/1 includes position 368 of the neuraminidase, in agreement with electron microscopy studies. "Docking" studies between the Fab and the neuraminidase are planned (35). Three Fab structures are currently under investigation in the authors’ laboratory. One of them is that of the anti-phenylarsonate mAbR19.9 (IgG2b, x), which does not carry the major idiotype of the mouse A/J strain (36). Another is from NQ10/12.5, an antiphenyloxazolone mAb (IgG1, x) (37). The third Fab is from the IgG1 (2) humanmyelomacryoimmunoglobulinHil (38). In these structures, the Fab fragments appear an extended conformation, with elbow angles close to 180° . Thus, it appears that the "elbow" angle does not correlate with a liganded or unliganded state of the antibody combiningsite in the different Fab structures that have been determined.

ANTIGEN-ANTIBODY

COMPLEXES

The introduction of cellular hybridization techniques for producing cell lines secreting antibodies of predefined specificity (39) openedthe wayfor the study of specific antibodies and antigen-antibody complexes by a variety of techniques, including X-ray crystallography which demands relatively large quantities of material (of the order of several hundred milligrams of antibody). X-ray crystallographic determinations of the structure of ligand-antibody complexes(reviewed in 1, 2) provided a structural model for binding reactions of antibodies. However, these studies

Annual Reviews

Annu. Rev. Immunol. 1988.6:555-580. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

560

ALZARI,

LASCOMBE & POLJAK

involved myeloma proteins and, mostlikely, cross-reacting ligands which becauseof their small size could only interact with part of the antigen combiningsite of the antibodies. Theligands used in those studies, phosphorylcholine and vitamin K~OH, bind with affinity constants, KA, of about 1 x l0 s M-1, which are below those observed for manyantigenantibody reactions. That no conformationalchangehad been observedin those ligand-Fab complexescould be attributed to limited interactions with the combiningsites. Other important questions remained to be answered. For example, does a common protein antigen unfold or otherwise changeits structure whencombinedto an antibody?Is the antigenic determinant(epitope) contiguous, or is it discontiguous, assembledfrom different parts of the primarysequencebrought together by the threedimensionalfolding of the macromolecularantigen? Howdo antigen variations give rise to cross-specificity or to lack of recognitionby specific antibodies? These questions can only be answered by the study of the threedimensionalstructure of an antigen-antibody complex.Several laboratories have intensively studied modelantigens using proteins of known three-dimensionalstructure. Theseoccur in cross-reactive, evolutionarily related formsin different organismsdisplayingsmall to large variations in aminoacid sequence. Among these protein antigens (reviewed in 40) we count cytochrome C, myoglobin, lysozyme, serum albumin, and the influenza virus neuraminidase.Thedetailed three-dimensionalstructures of three antigen-antibody complexesare nowknown,and they are reviewedin the followingparagraphs.

ANTILYSOZYME ANTIBODIES At least three laboratories havestudied in detail a large numberof monodonal antibodies against the model antigen hen egg-white lysozyme (HEL). Sercarz and collaborators (40, 41) have prepared more than distinct antibodies in the C57/BL strain of mice. Smith-Gilland colleagues (40, 42) and Harperet al (43) have inducedanti-HELmAbsin the BALB/c strain. Thesestudies havebeenprecededby those of Atassi et al (44) and of Mozeset al (45), Prager&Wilson(40, 46), etc. Sincethe workof Harper et al (43) and other subsequent, unpublishedworkhas been performed the laboratory of the authors of this review,it is summarized here. Morethan 40 anti-HEL mAbsobtained in the course of secondary responsesin BALB/c micehave beenstudied and characterized as part of a project attemptingto define antigen-antibodyinteractions at the molecular level by X-ray diffraction studies. All the anti-HELMAbsobtained were IgG1(x). Noneof themreact with humanmilk lysozyme.Bytheir reactivity

Annual Reviews

Annu. Rev. Immunol. 1988.6:555-580. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

STRUCTURE OF ANTIBODIES

561

patterns with eight different avian lysozymesthey can be divided into 11 groups.Theantibodieswithin each groupshare fine specificity, indicating that they recognizethe sameor closely adjacent antigenic determinants. For example, two mAbsthat belong to the same group, D1.2 and D1.3, share a reactivity pattern such that the replacementof Gln 121 in the HEL sequence(as in the lysozymesfrompartridge, turkey, etc.) results in the abolition of antibody binding to the antigen. Since D1.2and D1.3have different solubility properties(observedduringtheir purification andcrystallization trials), they are not identical. Similarly,mostof the antibodies included within a given group, differ fromeach other by physicochemical properties observedduring their preparation and fragmentationinto Fab and Fc. Theycould also differ in their precise recognitionspecificities. Consequently,the clustering into 11 groupsmayonly indicate the limitations of the bindingassaysbasedonthe restricted availability of different lysozymesequences. Additionalconclusionsof this study can be summarized as follows: (i) Theaffinity constants of the MAbsfor the HELantigen range from 8 × 106 -l. to 5 x 108 M (ii) Heteroclitic antibodiesbind heterologousantigens(other avianlysozymes)with affinity constants whichare somewhat higher although of the sameorder of magnitudeas those of HEL. (iii) Antibodiesrecognizethe antigenic surface of HELby close complementarity without producing long-range conformational changes on the antigen so that it can be simultaneously recognized by antibodies directed against different determinants. (iv) Althoughonly scattered points of the antigen’s surface that are recognizedby the antibodieshavebeenidentified it is mostlikely that they cover the wholeantigen’s surface. This conclusion is in agreementwith that obtained by several laboratories studying different antigen-antibody reaction(reviewedin Ref. 40), indicatingthat the entire surfaceof a protein is potentiallyantigenic. THREE-DIMENSIONAL OF A LYSOZYME-FAB

STRUCTURE COMPLEX

Using the anti-HEL mAbsmentioned above, a systematic search for crystallizing Fab-HELcomplexes was performed. The most favorable crystals for X-ray diffraction studies were obtained with antibody D1.3 whoseFab complexedto HELcyrstallized in the space group P21 with a = 55.6/~, b = 143.4/~, c = 49.1 A, fl = 120° and one moleculeof the complexin the asymmetricunit. The determination of the three-dimen-

Annual Reviews

Annu. Rev. Immunol. 1988.6:555-580. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

562

ALZARI,

LASCOMBE & POLJAK

sional structure of this complexat 6 ~ resolution and at 2.8/~ resolution has been reported (47, 48). The aminoacid sequences of the Vuand regions of Fab D1.3 were determined from the heavy and light chain complementaryDNAsequences (M. Verhoeyen, C. Berek, J. M. Jarvis, G. Winter, in preparation). The 562 aminoacid residues in the complex have been located in the electron density maps, and the model thus obtainedwasrefined to give a reliability factor R -- 0.28. Solventmolecules were not included in this model. Fab D1.3appears in a fully extended conformationin the complex,with a clear separation betweenthe variable (V) andconstant(Cri CL) doma ins. Its tert iary structure resembles that of other knownFabs, with the exceptionof its complementaritydetermining regions (CDR).Therelative positions of Vr~and VLare as in other Fab structures, indicating that no changein quaternary structure in the V domainis inducedby antigen binding. Specific changesin antibody conformation can only be detected by comparisonwith the unliganded Fab D1.3 structure, whichhas not yet beendetermined.However, the similarity with other Fab structures suggest that conformational changes in the complexedFab D1.3 should be small. A comparisonbetweenthe structures of native HEL(49) and that bound by Fab D1.3 gives a root meansquare deviation of 0.64 A at their 0~carbon(Ce) atoms.This overall differenceis not significant since the error in the Cepositions in the complexis about 0.6 ,~. Thelargest changes,up to 1.6/~, are seen in the region furthest awayfrom the antibody which showsdifferencesof the sameorder of magnitudewhendifferent crystalline forms of HELare compared. Formation of a complex with antibody D1.3 produces no more distortion of the structure of HELthan does crystallization underdifferent conditions. Thecontacts betweenantigen and antibodyare describedin Tables 1 and 2. Sixteen HELresidues form the discontiguous, conformationalepitope recognizedby D 1.3. Seventeenaminoacids fromthe antibody contact the epitope. The H chain contributes 10 of those residues, the L chain 7. All the CDRregions and, in addition, two residues that belong to the "framework"regions participate in contacts with the antigen, (see Table 1). The H chain makesmore contacts with the antigen than does the chain, and in particular, its third CDRloop makesmanymore contacts than any other CDR.VLCDR2contributes the least to antigen binding. Alarge numberof the antibodyside chains that contact the antigen (9 out of 17) are aromatic, presenting large areas of hydrophobicsurface to the antigen. Someof these chains (His30 and Tyr50in VL;Tyrl01in VH)also contribute to hydrogenbondingwith the antigen via their polar atoms. Thus, hydrogenbonds and van der Waalsinteractions describe the chemical nature of the antigen-antibody contacts. About750/~2(11%)of the

Annual Reviews STRUCTU"RE OF ANTIBODIES

563

Table 1 (a) Antibody residues involved in contact with lysozyme~, and (b) lysozyme residues ~ in contact with antibody (a)

Annu. Rev. Immunol. 1988.6:555-580. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Light chain CDR1 FR2 CDR2 CDR3

~ chain FR1 CDR1 CDR2

CDR3

Antibody residues Lysozyme residues in contact

His 30 Tyr 32 Tyr 49 Tyr 50 Phe 91 Trp 92 Ser 93

Leu 129 Leu 25, Gln 121, Ile 124 Gly 22 Asp 18, Asn 19, Leu 25 Gin 121 Gin 121, lle 124 Gin 121

Thr 30 Gly 31 Tyr 32 Trp 52 Gly 53 Asp 54 Arg 99 (96) Asp 100 (97) Tyr 101 (98) Arg 102 (99)

Lys 116, Gly 117 Lys 116, Gly 117 Lys 116, Gly 117 Gly 117, Thr 118, Asp 119 Gly 117 Gly 117 Arg 21, Gly 22, Tyr 23 Gly 22, Tyr 23, Ser 24, Asn27 Thr 118, Asp 119, Val 120, Gin 121 Asn 19, Gly 22

Lysozyme residues

Numberof antibody residues in contact

Asp 18 Ash 19 Arg 21 Gly 22 Tyr 23 Ser 24 Leu 25 Ash 27 Lys 116 Gly 117 Thr 118 Asp 119 Val 120 Gln 121 lie 124 Leu 129

1 L chain 2 H, L 1H 4 H(3), 2 H 1H 1L 1H 3 H 6 H 2 H 2 H 1H 5 H(1), L(4) 2L 1L

Sequencepositions are numberedas in Kabatet al (104) exceptfor VHCDR3,wherethe numbers Kabatet al (104) are givenin parenthesis. Reproduced from reference 48, with permissionfrom Science magazine.

Annual Reviews

564

ALZARI, LASCOMBE& POLJAK

Table 2 Hydrogenbondedinteractions betweenantibody and lysozyme Antibodyresidue

Lysozymeresidue

N,2 His 30 O, Tyr 50 O Phe 91

O Leu 129 O~ Asp 18 N,2 Gln 121"

Or~Thr 30 N Gly 31 N Gly 53 N,~ Arg99 (96) O~Asp I00 (97) O~2Asp100 (97) O~Tyr I01 (98) O, Tyr 101 (98) O, Tyr 101 (98)

O Lys 116" O Lys 116 O Gly 117’ O Gly 22 N~2Asn 27 Or Ser 24* N Val 120 N Gin 121 O~ Asp 119

Light chain

Annu. Rev. Immunol. 1988.6:555-580. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Heavychain

*Denotes theclosestinteractions(distances < 2.5 ]k) Sequencepositionsare numbered as in Kabatet al (104) exceptfor Vr~CDR3 wherethe numbers Kabatet al (104)are givenin parenthesis. (Reproduced fromreference48, withpermission fromSciencemagazine.) solvent accessible surface of HELand about 700 A2 of the Fab are buried by complex formation. It has been proposed (50) that the calculation of the free energies association for the FabD1.3-HEL complex indicates the possibility of conformational changes after complex formation. However, a reliable calculation of the free energy of association for such complex is difficult if not impossible due to uncertainties inherent in the estimation of different contributions. Leaving these uncertainties aside, a different estimate could be obtained in general agreement with the observed association constant, as follows. An empirical, overall value of the hydrophobic contribution to the free energy (AG) of complex formation can be obtained from the area of the antigen-antibody interface excluded from solvent contact, assuming that little or no variation in conformation occurs after complex formation. In the FabD1.3-HEL complex these areas are of about 750 ,~2 for the antibody combining site and of about 700 A2 for the antigen. Thus, assuming an average value of 20 calories mol- 1 per A2 of buried surface area (51), we have 1450/~2 × 20 cal mo1-1 ~-2 or about 30 Kcal mol-~ for the free energy of complex formation. Since the intrinsic dissociation constant for the FabD1.3-HEL complex is KD = 2 × 10 -8 M, the free energy of dissociation GD= RTlnKI~ is of about 10 Kcal. Unfortunately, the calculation of the rotational and translational entropies lost as a result

Annual Reviews

Annu. Rev. Immunol. 1988.6:555-580. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

STRUCTURE OF ANTIBODIES

565

of complex formation is less straightforward. A proposed calculation for interacting proteins (51) suggests that their addedvalue is about twice that of the free energy of dissociation. If we assumethat this wouldalso be the case for the FabD1.3-HELcomplex, the sum of the free energies of complex formation would give a value in good agreement with the solvent entropy. At any rate, it is clear that these rough estimates cannot be used to postulate conformational changes (50) as a way of dissipating excess energies obtained by complex formation. Changes of conformation are more readily observed by a direct comparison of the experimentally determined structures of the free antigen and antibody with those of their liganded forms. Somatic recombination and imprecise joining of the VH-DH and DH-JH genetic segments of the H chain and of the L chain generate antibody diversity affecting position 96 in x chains and CDR3in H chains (52, 53). The structural model of Fab D1.3-HELallows an evaluation of the contribution of that diversity to antigen binding. VLresidue Arg96 is relatively distant (> 4/~) from the antigen and so are the J~-encoded residues. The residues encoded by the D segment of CDR3,Arg99, Asp 100, Tyrl01, and Argl02 make very specific contacts with the antigen. By contrast, neither JH residues nor those that could originate from imprecise joining at the DH-JHjunction contribute directly to the contacts made by antibody D1.3 with its antigen. A study (54) of the germline affinity ofanti5-dimethylaminonaphtalene-l-sulfonyl (DNS) IgM antibodies suggested that the affinity and selectivity of the IgMantibodies is primarily associated with the CDR3regions. The overall picture of the antigen-antibody interface in the Fab D1.3HELcomplex is that of two irregular, rather fiat surfaces with protuberances and depressions that fit into the complementaryfeatures of the other. The "lock and key" metaphor adequately describes this interaction and the fact that antigen and antibody form a complex without major conformational change. The tightness of the antigen-antibody interface explains in general why variants of the antigen are not recognized by antibody D 1.3. For example, avian lysozymes,as in the case of partridge and California quail lysozymes, with a Hisl21 residue instead of the Glnl21 present in HELare not recognized by D1.3, (43, 47, 48). Japanese quail lysozyme has an Asnl21 instead of Glnl21, but this difference alone would seem too small to explain the fact that antibody D 1.3 does not bind this lysozyme(KAabout 1 x 104 M-1). However,there are other sequence differences with HELat positions that make contact with the antibody: Asnl9 ~ Lys and Arg21 ~ Gin.

Annual Reviews

566

ALZARI,

LASCOMBE & POLJAK

Annu. Rev. Immunol. 1988.6:555-580. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

A NEURAMINIDASE FAB COMPLEX The three-dimensional structure of another antigen-Fab complex has been reported (55). In this crystalline complex,the antigen is the avian influenza virus neuraminidase of subtype Ng, and the Fab is derived from a murine antineuraminidase mAb(IgG2a, ~), NC41. The neuraminidase is a tetrameric membraneprotein of molecular weight 60,000 whose three-dimensional structure has been previously determined (56). In the complex, its surface loops at positions 368-370, 400-403,430-434, and 325-350 contact the CDRsof Fab NC41. The conformation at positions 325-350 has not yet been firmly established; it could be a region subject to extremethermal motion whose structure is difficult to determine even in the unliganded neuraminidase. The CDR3of the H and of the L chain have not yet been traced in the electron density maps so that a complete description of the antigen-antibody interface is not possible. However,the authors (55) estimate that there maybe 16 or 17 contacting residues, as in the Fab D 1.3-HEL complex described above. All the CDRsmake contact with the antigen with the possible exception of CDR1of the L chain. The complexed Fab NC41has an elbow angle of about 150°. A comparison of the relative positions of the VHand VLdomainsof the structure suggested to the authors that there is a small sliding movementof one domainrelative to the other (of about 1 ,~) and a rotation such that the distances between CDRsof the H and of the L chain are altered by 340 +/~ compared to those of the humanimmunoglobulins Newand Kol and the murine immunoglobulin McPC603.This "conformational" change in quaternary structure is postulated to result from complexformation with the antigen. The loop including residues 367-371 of the neuraminidase antigen was remodelled. The Ca atoms at positions 368-371 were movedby about 1/~ from their corresponding positions in the uncomplexed neuraminidase. This difference is interpreted by the authors as a distortion of the antigen induced by complex formation with the antibody. Furthermore, they claim that this interpretation is in agreement with the fact that antibody NC41 inhibits the neuraminidase activity against a small substrate, the trisaccharide sialyl-lactose. It is suggested that a displacement of Arg371is responsible for enzymeinactivation since directed mutagenesis experiments show that replacement of that residue by Lys results in the loss of 90%to 95%of enzyme activity. Since the crystallographic study of the neuraminidase-FabNC41is still preliminary, an evaluation of its results seems to us premature. It would be interesting to compare more immunoglobulin Fab fragments to see if the alleged difference in the Vn-VLpairing is really characteristic of Fab

Annual Reviews STRUCTURE OF ANTIBODIES

567

NC41. Equally important would be to study its structure in the uncomplexed state and to compare it with that observed in the complex. Concerning the proposed changes in the conformation of the antigen, similar displacements have been observed in the HELcomplexed to Fab D1.3, albeit in a region remote from antibody contacts, a region that shows conformational variability when different crystalline lysozymes are compared.

Annu. Rev. Immunol. 1988.6:555-580. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

OTHER LYSOZYME-FABCOMPLEXES The three-dimensional structure of a third antigen-antibody complex has been determined: that of the Fab Hy HEL-5complexed to lysozyme (57). Immunochemical studies had already indicated (58) that the BALB/c murine mAbHyHEL-5(IgG1, x) binds to a region of HELthat includes residue Arg68. This structure has been determined to a resolution of about 2.6 A and subsequently refined. A large surface of lysozyme (about 12% of its total surface) is in contact with the antibody. All CDRsof the antibody as well as VnTrp47, a "framework"residue, contact the antigen. There are 14 directly contacting residues of lysozyme at positions 41 to 53, 67 to 70, and 84. They are Gin41, Thr43, Asn44, Arg45, Asn46, Thr47, Asp48, Gly49, Tyr53, Gly67, Arg68, Thr69, ProT0 and Leu84. The L chain provides 7 directly contacting residues: Asn31, Tyr32, Asp40, Trp91, Gly92, Arg93 and Pro95. The H chain contributes 10 directly contacting residues: Trp33, Glu35, Trp47, Glu50, Ser54, Ser56, Thr57, Asn58, Gly95 and Tyr97. Thus, the epitope is noncontiguous or conformational. Arg68 and Arg45 which form a hydrogen-bond in native HELappear in a similar orientation in the complex, forming a ridge on the surface of the antigen which fits into a groove of the antibody, where they makesalt linkages to Glu35 and Glu50 of the H-chain. In bobwhite quail lysozyme, residue 68 is Lys; this replacementdrastically diminishes the association constant with HyHEL-5,in general agreement with the structural model. The relative orientation of the VH, VLdomains is the same as in other Fabs. In the complexed HEL, Pro70 which is part of a highly mobile region of the polypeptide chain appears to have movedby about 1.5 A from its position in the uncomplexedHEL.Another interesting feature of this complex is its crystalline polymorphism.This is detected experimentally by a range of values for the b axis of the unit cell of the crystals going from 65.2 to 74.8 ~. The determination of the structure of two of these crystalline forms reveals that they possess slightly different elbowangles. This observation further supports the conclusion that antigen binding does not trigger extension or contraction of the Fab structure. A comparison of the structures of the D1.3-HEL and the HyHEL-5

Annual Reviews

Annu. Rev. Immunol. 1988.6:555-580. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

568

ALZARI,

LASCOMBE & POLJAK

complexes reveals commonfeatures such as similar areas of interaction, similar number of contacting residues, small conformational changes in the antigen and presumably in the antibody, participation of all the CDRs and of one or two residues of the frameworkregions in the contacts with antigen. In both cases, the identification of aminoacid residues included in the epitope by immunochemical methods (43, 58) was confirmed the three-dimensional crystal structure analysis. However,there are some differences such as the presence of electrostatic interactions in the HyHEL5 complex with HEL,absent in the D1.3-HELcomplex. In this context it is interesting that the affinity constant of the HyHEL-5 complex(2.5 × l09 M-1) appears to be about two orders of magnitude higher than that of D1.3-HELcomplex(4.5 × l07 M-1). Another interesting difference resides in the fact that in D1.3-HELthe larger numberof contacts is madeby Vn, CDR3whereas in HyHEL-5it is VL CDR1and VH CDR2that make the larger numberof contacts with the antigen. The structure of the Fab fragment from a heteroclitic monoclonalantiHELantibody complexed with a heterologous antigen, pheasant lysozyme, is under study in the authors’ laboratory (59). Fromcross-reactivity studies (43), the epitope is expected to include Asnl9, Arg21, and/or Glyl02, Asnl03 near the enzymeactive site. This epitope does not coincide with those recognized by antibodies D1.3 and HyHEL-5. The three-dimensional structure of a complex between HELand a specific mAbcombining site has been proposed by Rees and colleagues (60). This antibody, Gloop 2, was raised against a peptide containing the "loop" determinant of HEL(residues 57-84), and it is also capable binding native HELwith an affinity constant, KA, of 4 x 106 M-~ which is about one order of magnitude below that for the loop peptide. In the first step the authors modeled the structure of the VHand VLregions of the antibody on the basis of knownthree-dimensional structures of Fab fragments and L-chain dimers. The model was subsequently submitted to energy minimization to improve its stereochemistry. By "docking" this model of the antibody combining site with the tentatively characterized antigenic determinant on HEL,a model of the complex was generated. In this hypothetical model the interacting surface measures 20 x 15 A, with all CDRsin contact with the antigen. In contrast with the HEL-Fab D1.3 complex, a conformational change was introduced in the antigenic determinant. Further refinement of this calculated model was recently carried out by energy minimization (61) leading to a more complementary antigen-antibody interface with only minor conformational changes, in agreement with the observations on the HEL-Fab D1.3 complex. The model implicates interactions between VL GIu28 with HELArg68 and VH Lys56 with HELAsn77. Site directed mutagenesis experiments showed

Annual Reviews

Annu. Rev. Immunol. 1988.6:555-580. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

STRUCTURE

OF ANTIBODIES

569

that the nature of these residues is indeed importantin antigen binding, since a double mutant Glu28 ~ Ser, Lys56 ~ Gln showed an eight to ninefold increase in affinity for HELand a four to fivefold increase for the loop peptide. Furthermore, the authors conclude that removal of electrostatically chargedresidues and their replacementby non-charged residues with hydrogen-bondpotential allows formation of additional hydrogenbondsbetweenantigen and antibody without destabilizing interactions within the CDRsof the antibody at the mutationsites. Replacement of VHGlu50by Ser resulted in complete loss of binding between Gloop2and HELor loop peptide. Themodelassigns this residue to a salt linkage interaction with VHArgl01and a hydrogenbondto Tyr94in VL. It thus predicts that substitution of Glu50will causea loss of stabilizing interactions within CDRs,leading to a loss of binding, as observedin the site directed mutagenesisexperiment. Althoughthe proposedstructural modelhas successfully guidedexperimentsand explainedbinding phenomena, its intrinsic accuracystill needsto be verified MAPPING

ANTIGENIC

DETERMINANTS

Mappingof antigenic determinants (or epitopes) by biochemicaltechniques has been proposed. Oneof these procedures (62) involves proteolysis of antigen-antibodycomplexes.The rate of proteolysis will be lower for the antigenic determinant whichis protected by the antibody and thus identified. Using two murinemAbsspecific for cytochromec, trypsin proteolysis indicated binding of the monoclonalantibodies to conformationalepitopes. Theauthors observedthat not all of the antigen that is protectedfromproteolysis is necessarilyin direct contact with the antibodycombining site, since it couldalternatively be protectedby steric hindrance. In addition, not all antigens are susceptible to experimental proteolysis. However,manydifferent proteolytic enzymescould be used to overcome this difficulty. Anotherproposedprocedure for epitope mappinginvolves chemicalmodification of an antigen in its free and antibody-boundstates (63). The application of this techniqueto cytochrome c is inspired by its successful use in mappingmolecularinteractions ofcytochrome c with redoxproteins. Therelative rates of acetylation of lysines and threonines weremeasured in the presence or absence of an antihorse cytochromec antibody. Two lysine residues, Lys60and Lys99,werethus implicatedin interactions with the antibody. In suitable cases, the two approachesoutlined above may extend the informationobtainedby direct binding studies of evolutionary or chemicalvariants of suitable antigens. Inducedvariations of the antigenie determinant by site directed mutagenesiscould be used as a sys-

Annual Reviews 570

ALZARI,

LASCOMBE & POLJAK

tematic wayof exploring the extent and nature of the antigen-antibody interactions.

Annu. Rev. Immunol. 1988.6:555-580. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

ANTIGENICITY Thenotion ofantigenicity of a given macromolecule has to be differentiated from that of its immunogenicity.Antigenicity is the property of being recognized by a molecule of the immuneresponse, usually an antibody. Immunogenicity refers to the ability to induce an immune response and is the result of the properties of the immunogen as well as those of the immunesystem of the animal challenged by the immunogen and of the procedurefollowedfor that challenge. Anantigenic determinantor epitope is that part of the antigen that interacts with or is ligandedby a B-cell receptor and subsequently, in the immuneresponse, by antibodies. The antigenic determinantor epitope of a native macromolecule is locatedon its surface. Bycontrast, determinantsthat are recognizedby T-cell receptors, usually in conjunctionwith (self) histocompatibilityantigens, arise from proteolytic processing of the antigen and are frequently located in the inner, unexposed parts of its native structure. Antigenicdeterminantshavetraditionally been divided into two structural categories: (a) continuousdeterminants,in whichall the residues contact with antibodyare containedwithin a single segmentof the aminoacid sequence of the antigen and (b) noncontinuousor "topographical" determinants, in whichresidues are far apart in the sequencebut are broughttogether by the folding of the protein in its native conformation. Theprobability of occurrenceof each of these types of determinantsin native proteins dependson what proportion of the surface of a globular protein is madeup of linear arrays of residues. Barlowet al (64) have calculated this proportion as a function of increasing radius of the recognition site. Even whenthe contact radius is as small as 6 ,~, only approximately 25%of the surface is continuous; with a more realistic radius of 12.5 ,~, as in the three-dimensionalstructures of the antigen-antibodycomplexesstudies thus far, virtually none of the surface is continuous. Therefore most antibodies directed against native protein molecules probably recognize noncontinuousdeterminants. Studies of the antigenic determinantsrecognizedby antisera led Atassi to postulate an "antigenic structure" for myoglobin (65) and lysozyme(44), consisting of few epitopes whichwouldbe recognized by the polyclonal antibody molecules of a typical immuneresponse in the experimental animal,the rabbit. This is evidentlya schematic,controversial conclusion since an immuneresponse is influenced by such complexfactors as the methodand site of immunization,dose, nature of the immunogen, presence

Annual Reviews

Annu. Rev. Immunol. 1988.6:555-580. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

STRUCTURE OF ANTIBODIES

571

or absence of adjuvants, genetic endowment of the test animal and its physiologicalcondition, etc. This proposalhas metwith extensivecriticism (see 40). Thusfor example,Wilsonand colleagues were able to showthat a rabbit antiserum could detect the antigenic variations present in the evolutionarily related lysozymesformegg-whitesof chickenand partridge or quail (66). These experiments showedthat a determinant including position 121 in the lysozymesequencecould be detected by antibodies. This determinantdoes not correspondto the postulated "antigenic structure" (44) of lysozyme.Sela (67) had earlier proposedthat antiprotein antibodies bind relatively short polypeptidechain segmentsof the protein. Consequently,antiprotein antibodies could be inducedby synthetic peptides adequatelyconjugatedto a carrier as shownby experimentsusing a "loop" peptide (23 aminoacids) of lysozyme(68), a peptide of the protein of the bacteriophageMS2(69), a syntheticpeptide of the diphtheria toxin (70) and others. This idea has received a renewedimpetus(71) herald of newvaccines and newways of manipulating immuneresponses for therapeutic purposes. However,antipeptide antibodies will often not recognizethe native protein fromwhichthe peptides are derived unless it is denatured.Thosereactions that are observed,often showa loweraffinity and less discrimination than those observedwith antibodies against the native protein. PREDICTION

OF ANTIGENIC

SITES

Criteria for the predictionof protein antigenicityas well as it correlation with different structural features of proteins suchas hydrophilicity (72, 73), "immunogenic potential" (74), mobility (75, 76), and accessibility (77-79) have been vigorously discussed and frequently reviewed(see example(40, 55, 80-87).If weconsiderthat in the case of lysozyme, specific antibodies contact about one ninth of the molecularsurface (48, 57), and that at least 10 different fine specificities can be detected evenwith an incompletepanelof antigenicvariants (43), it followsthat essentially the entire surfaceis recognizableby antibodiesirrespective of its hydrophobic, hydrophilic, or mobility characteristics. In this sense accessibility is a minimaland, webelieve, sufficiently general condition to define an antigenic determinant. Mappingof complete antigenic determinants has only recently been achievedby X-raycrystallographicstudies. Althoughdecisive, these studies are not yet sufficiently easy to performin order to further clarify the structural correlation of antigenicity in the manyantigen-antibodysystems that have been explored by immunochemical studies. In the meantime, prediction of antigenic sites of a protein can be helpedonly in a general

Annual Reviews

572

ALZARI,

LASCOMBE & POLJAK

way by a knowledgeof its detailed three-dimensional structure, showing which parts of the amino acid sequence are exposed on the surface of the molecule.

Annu. Rev. Immunol. 1988.6:555-580. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

CONFORMATION OF IDIOTYPES Idiotypes (88, 89) can be defined as antigenic determinants unique to given antibody molecule. Althoughthe serological bases ofidiotype detection introduce somerelativity into that idealized definition, idiotypes have becomean important subject of research, recently reviewed in this series (90). The reader is referred to that review for an in-depth treatment of the topic. Direct correlation of three-dimensional structure and idiotypy has so far only been achieved by immuno-electron microscopy studies (91, 92). These have shownthat an antigen-inhibitable idiotype is localized in the distal terminus of Fab arms, as might be expected from immunochemical data. Other, less private idiotypes (IdX), as well as allotypes, were shown by the same technique to be located in different parts of the Fab arms including the constant CL and C~I domain. The relatively low resolution of these studies does not allow a more precise definition. By analogy with the interactions observed in antigen-antibody complexes it can be postulated that a combining-site related idiotope will consist of part of VL and Vn adding up to 15 to 20 amino acids of the CDRregions and "framework"residues combined. It is likely that the interaction between the idiotope and its anti-idiotope will be one between two closely complementary, irregular, but rather flat surfaces in close association, as in an antigen-antibody complex (48). A preliminary X-ray diffraction study of a complexbetween an idiotope and an anti-idiotope has been reported (93). The idiotope-carrying Fab that from the anti-HEL mAbD1.3 reviewed above; the anti-idiotope Fab is from the mAbE225, produced in the same (BALB/c) strain of mice D1.3. Formation of the complex is inhibited by the HELantigen for which the idiotope-carrying FabD1.3 is specific. This study should eventually allow the structural definition of an idiotope and its relation to the antigen contacting residues of the antibody combiningsite.

TECHNICAL APPLICATIONS SPECIFICITY

OF ANTIBODY

Specific antibodies are widely used as analytical tools in biology and medicine. Cell hybridization techniques (hybridomas) have vastly

Annual Reviews

Annu. Rev. Immunol. 1988.6:555-580. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

STRUCTURE OF ANTIBODIES 573 increased the analytical and technical applications of mAbswhichare too numerousto be reviewed here. However,we would like to mention two developmentswhichare conceptually interesting and potentially useful: (a) the grafting of specific CDR regions onto frameworksof other antibodies; (b) the possible use of antibodies to catalyze chemicalreactions. RecombinantDNA techniques have been used for the genetic engineering of antibodies (94-96) and for grafting murine CDRregions of known specificity onto frameworkregions of humanantibodies of different classes, thus producing chimeric, "humanized"antibodies. For example, the Hchain CDRsof a murineanti-4-hydroxy-3-nitrophenylacetylcaproic acid mAb(B1-8) were grafted onto the H chain of a humanmyeloma protein. This chimeric H chain recombinedwith the mouseB1-8L chain gave a mAbwith the hapten affinity of the B1-8antibody (97). Thus, chimeric antibodies consisting of humanconstant and variable domains can be preparedwhichare specific for selected antigens and whichcould have therapeutic value. These experiments can be combinedwith site directed mutagenesisto explore the role of different aminoacid residues in determiningthe specificity of the antibodycombining site. Antibodies, like enzymes,can specifically bind a chemicalsubstrate although they will not hydrolyzeit. However,it wassuggested(98) that specific antibodies that bind transition state analogs could catalyze a chemicalreaction. This has been demonstratedto be the case in several laboratories. Kohenet al (99) demonstratedthat mAbsagainst DNPare able to enhancethe hydrolysis of a DNP-e-aminocaproyl-coumarin ester. Tramontano et al (100) showedthat mAbsspecific for a phosphonateester catalyze the hydrolysisof analogouscarboxylicesters. Pollacket al (101) using the nitrophenyl phosphorylcholinebinding mousemyelomaprotein MOPC167 also observed the hydrolysis of an analogous carbamate. In spite of the fact that these catalytic antibodies behavelike enzymes in showingcompetitiveinhibition, substrate specificity, and saturation kinetics, their catalytic rates are modest,only about 1000-foldabovethat of the uncatalyzedreaction, and about 5,000- to 10,000-foldbelowthat of an esterase. In a morerecent report (102) a mAbaccelerated the rate hydrolysis of methyl 4-nitrophenyl carbonate by a factor of 16,500over that of the uncatalyzed reaction. Further developmentsin the use of catalytic antibodiesfor the synthesisof different productscan be expected in the future. However,.it should be noted that manylaboratories are designingand synthesizing organic compounds that possess similar enzyme activities. Althoughin these experimentsthe rate accelerations obtained surpass those of chymotrypsin,they all use esters, in particular p-nitrophenylesters, as substrates. Theseesters are very reactive, and they are rather artificial substrates(103).

Annual Reviews

574

ALZARI,

LASCOMBE & POLJAK

Annu. Rev. Immunol. 1988.6:555-580. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

PREDICTION OF ANTIBODY STRUCTURES As reviewed before (1, 2) and above, antibodies display a constant polypeptide chain folding pattern, the immunoglobulin-fold (15, 16) for the hypervariable (104) CDRloops of H and L chains. This observation has prompted repeated attempts to predict the detailed structure of antibody combining sites based on their amino acid sequences and homology(60, 105-109). In these cases there could be no evaluation of the proposed models based on a comparison with an experimentally determined structure, with the exception of the Fab J539 discussed in a preceding section. Interesting speculations are those in which immunoglobulin structures are used as a basis for the modelling of evolutionarily related but less homologousproteins such as the major histocompatibility antigen (110) or the T cell antigen receptor (111). As observed by Chothia & Lesk (112) and Blundell and colleagues (113), the average accuracy of positioning amino acid residues increases with the homology between the modeled protein and the one used as the base model. Snow& Amzel (114) using "coupled perturbation procedure" calculated the detailed three-dimensional structure of Fab Kol using Fab Newas their data base. The Fab Kol structure thus obtained closely agrees with that found experimentally. Basically, the procedure consists of a search of low-energy conformations for each side chain that is introduced and for the neighboring side chains likely to be affected by the substitution; the lowest energy structure is considered to be the correct model. Chothia et al (115) have predicted the structure of the combiningsite Fab D1.3 and comparedit with the determined cyrstalline structure. The predicted model was based: (a) on a comparative analysis with known antibody structures which provided a starting model for most CDR regions; and (b) on energy calculations which indicated the most likely lowenergy conformations. Comparison with the experimentally determined model indicated that VL CDR1, CDR2and VH CDR2, CDR3 were predicted in a satisfactory way (average root’mean square deviation of about 0.7/~). Since the predicted and the experimentally determined structures agree reasonably well, the authors conclude that the association with antigen does not induce a significant change in the conformation of the antibody.

ACKNOWLEDGMENTS Wethank D. R. Davies, P. Saludjian, F. A. Saul, and P. Tougard for communicatingresults prior to publication.

Annual Reviews STRUCTURE OF ANTIBODIES

575

Research work in the authors’ laboratory is supported by grants from CNRS, Institut Pasteur and contract BAP-0221(DC) from the EEC.

Annu. Rev. Immunol. 1988.6:555-580. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Literature Cited 1. Amzel, L. M., Poljak, R. J. 1979, Three-dimensional structure of immunoglobulins. Ann. Rev. Biochem. 48:961-97 2. Davies,D. R., Metzger,H. 1983.Structural basis of antibodyfunction. Ann. Rev. Immunol.I: 87-117 3. Rajan, S. S., Ely, K. R., Abola,E. E., Wood,M. K., Colman,P. M., Athay, R. J., Edmundson, A. B. 1983. Threedimensionalstructure of the McgIgG1 immunoglobulin. MoLlmmunol. 20: 797-99 4. Sarma,V. R., Silverton, E. W.,Davies, D. R., Terry, W.D. I971.~Thethreedimensionalstructure at 6 Aresolution of a humanyG1immunoglobulinmolecule. J. Biol. Chem.246:3753-59 5. Matsushima,M., Marquart, M., Jones, T. A., Colman,P. M., Barrels, K., Huber, R., Palm, W.1978. Crystal structure of the humanFab fragment Kol and its comparisonwith the intact Kol molecule. J. Mol.Biol. 121: 44159 6. Ely, K. R., Colman,P. M., Abola, E. E., Hess, A, C., Peabody,D. S., Parr, D. M., Connell, G. E., Laschinger,C. A., Edmundson, A. B. 1978. MobileFc region in the Zie IgG2cyroglobulin: comparisonof crystals of the F(ab’)~ fragment and the intact immunoglobulin. Biochemistry17:820-23 7. Mariuzza,R. A., Poljak, R. J., Mihaesco, C., Mihaesco,E. 1983.Crystals of the humanheavychain disease protein Riv and humanFc fragment are isomotphous:further evidence for conformational flexibility in the hinge regions of immunoglobulins.J. Mol. Biol. 165:559-61 8. Humphrey,R. 1986. Computermodels of the humanimmunoglobulins:shape and segmental flexibility. Immunol. Today 7:174-78 9. Gregory,L., Davis, K. G., Sheth, B., Boyd,J., Jeffers, R., Nave,C., Burton, D. R. 1987. The solution conformationsof the subclassesof humanIgG deducedfrom sedimentation and small angle X-rayscattering studies. Mol. Immunol. 24:821-29 10. Oi, V. T., Vuong,T. M., Hardy, R., Reidler, J., Dangl,J., Herzenberg,L.

A., Stryer, L. 1984. Correlation between segmental flexibility and effector functionof antibodies. Nature 307:136-40 11. Wringley,N. G., Brown,E. B., Skehel, J. J. 1983. Electron microscopicevidencefor the axial rotation andinterdomainflexibility of the Fab regionsof immunoglobulinG. J. MoLBioL 169: 771-74 12. Roux, K. H. 1984, Direct demonstration of multiple VHallotopes on rabbit Ig molecules:allotope characteristics andFabarmsrotational flexibility revealed by immunoelectron microscopy.Eur. J. Immunol.14: 45964 13. Padlan, E. A., Cohen,G. H., Davies, D. R. 1987. Studiesof the tertiary and quaternary structure of antibody constant domains.In Bioloyical Organization: Macromolecular Interactions at Hitlh Resolution, ed. R. M. Burnett, H. J. Vogel. NewYork: Academic.pp. 193-214 14. Deisenhofer,J. 1981.Crystallographic refinement and atomic models of a humanFc fragment and its complex with fragment B of a protein A from Staphylococcus aureusat 2.9 and2.8/~ resolution. Biochemistry20:2361-70 15. Poljak, R. J., Amzel,L. M., Avey,H. P., Chen, B. L., Phizackerly, R. P., Saul, F. 1973.Three-dimensional structure of the Fab’ fragmentof a human immunoglobulin at 2.8 /~ resolution. Proc. Natl. Acad. Sci. USA70: 330510 16. Schiffer, M., Girling,R. L., Ely,K. R., Edmundson,A. B. 1973. Structure of a 2-type Bence-Jonesprotein at 3.5/~ resolution. Biochemistry12:4620-31 17. Chothia, C., Novotny,J., Bruccoleri, R., Karplus, M. 1985. Domainassociation in immunoglobulinmolecules; The packing of variable domains. J. Mol. Biol. 186:651-63 18. Novotny,J., Bruccoleri, R., Newelle, J., Murphy,D., Haber,E., Karplus,M. 1983. Molecularanatomyof the antibodybindihg site. J. Biol. Chem.258: 14433-447 19. Allison,J. P., Lanier,L. L. 1987.Structure, functionandserologyof the T-cell

Annual Reviews 576

ALZARI, LASCOMBE& POLJAK

Annu. Rev. Immunol. 1988.6:555-580. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

antigen Immunol.receptor 5:503-40complex.Ann. Rev. 20. Becker,J. W., Reeke,G. N. Jr. 1985. T~ree-dimensional structure of fl2-microglobulin. Proc.Natl. Acad.Sci, US.4 82:4225-29 21. Mostov, K. E., Friedlander, M., Blobel,G. 1984.Thereceptor for transepithelial transport of IgA and IgM contains multiple immunoglobulin-like domains. Nature 308:37-43 22. Williams, A. F., Gagnon, J. 1982. Neuronal cell Thy-I glycoprotein: homologywith Ig. Science 216: 696703 23. Clarck, M.J., Gagnon, J., Williams,A. F., Barclay, A. N. 1985. MRCOx-2 antigen: a lymphoid/neuronal membraneglycoproteinwitha structure like a single immunoglobulin light chain. EMBO.L 4:113-18 24. Ishioka, N., Takahashi, N., Putnam, F. W.1986. Aminoacid sequence of humanplasma ~lB-glycoprotein: homologyto the immunoglobulinsupergenefamily. Proc.Natl. Acad.Sci. USA 83:2363-67 25. Cunningham, B. A., Hemperley,J. J., Murray,B. A., Prediger, E. A., Brackenbury, R., Edelman, G. M. 1987. Neural cell adhesionmolecule:structure, Ig-like domains,cell surface modulationand alternative RNA splicing. Science 236:799-806 26. Williams,A. F., Barclay, A. N. 1988. The immtmoglobulin superfamily-Domainsfor cell surface recognition. Ann. Rev. lmmunol. 6:000~0 27. Sub, S. W., Bhat, T. N., Navia,M.A., Cohen,G. H., Rao, D. N., Rudikoff, S., Davies, D. R. 1986. The galactanbinding immunoglobulinFab J539: an X-raydiffraction study at 2.6 /~ resolution. Proteins 1:74-80 28. Glaudemans,C. P. J., Kovac,P. 1985. Probing the combiningsite of monoclonal IgA J539 using deoxyituoroand other galactosidesas ligands. Mol. Immunol. 22:651-53 29. Glaudemans,C. P. J., Kovac,P., Rasmussen,K., 1984. Mappingof the subsites in the combiningarea of monoelonal antigalactan IgA J539. Biochemistry 23:6732-36 30. Satow, Y., Cohen,G. H., Padlan, E. A., Davies, D. R. 1986. Phosphocholine binding immunoglobulin Fab McPC603.An X-ray diffraction study at 2.7 ,~ J. Mol.Biol. 190: 59331. Chang,C. H., Short, M. T., Westholm, F, A., Stevens, F. J., Wang,B.-C., Furey, W.Jr., Solomon,A., Schiffer,

32.

33.

34.

35.

36.

37.

38.

39.

40.

M. 1985. Novel arrangement of immunoglobulinvariable domains:Xray crystallographicanalysis of the 2chain dimer Bence-Jonesprotein Loc. Biochemistry 24:4890-97 Chang,C. -H., Carperos, W.E., Ainsworth,C. F., Olsen,K.W.,Schiffer, M. 1987.Pronounced effect of the solvent of crystallization on the structure of a multidomainprotein. Acta Cryst. A. (Suppl.) 43:C-19 Cygler, M., Boodhoo,A., Lee, J. S., Anderson,W.F. 1987. Crystallization andstructure determinationofan autoimmune anti-poly(dT) immunoglobulinFab fragmentat 3.0 .~ resolution. J. Biol. Chem.262:643 ¯ Gibson,A. L., Herron,J. N., Ballard, D. W., Voss, E. W.Jr., He, X. M., Patrick, V. A., Edmundson, A. B. 1985. Crystallographic characterization of the Fab fragmentofa monoclonalantiss-DNAantibody. Mol. Immunol. 22: 499-502 Colman,P. M., Webster, R. G. t987. Thestructure of an antineuraminidase monoclonalFab fragmentand its interactionwiththe antigen.SeeRef. 13, pp. 125-33 Mariuzza,R. A., Amit,A. G., Boulot, G., Saludjian,P., Saul, F. A., Tougard, P., Poljak, R. J., Conger,J., Lamoyi, E., Nisonoff, A. 1984. Crystallization of the Fab fragments of monoelonal anti-p-azophenylarsonate antibodies and their complexeswith haptens. J. Biol. Chem.259:5954-58 Mariuzza,R. A., Boulot, G., Guillon, V., Poljak, R. J., Berek,C., Jarvis, J. M., Milstein, C. 1985. Preliminarycrystallographic study of the Fab fragments of two monoclonalanti-2-phenyloxazoloneantibodies. J. BioL Chem. 260:10268-70 Humphrey,R. L., Avey,H. P., Becka, L. N., Poljak, R. J., Rossi,G., Choi,T. K., Nisonoff, A. 1969. X-ray crystallographic study of two Fab fragments from the humanmyelomaproteins. J. MoLBioL 43:223-26 Kthler, G., Milstein, C. 1975. Continuouscultures of fused cells secreting antibody of predefined specificity. Nature 256:495-98 Benjamin,D. C., Berzofsky,J. A., East, I. J., Gurd, F. R. N., Hannum,C., Leach, S. J., Margoliash,E., Michael, J. G., Miller, A., Prager,E. M., Reichlin, M., Serearz,E. E., Smith-Gill, S. J., Todd, P. E., Wilson, A. C. 1984. The antigenic structure of proteins: a reappraisal. Ann. Rev. Immunol.2: 67101

Annual Reviews

Annu. Rev. Immunol. 1988.6:555-580. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

STRUCTUREOF ANTIBODIES 41. Miller, A., Ch’ng,L.-K., Benjamin,C., Serearz, E., Brodeur, P., Riblet, R. 1983. Detailed analysis of the public idiotype of anti-hen egg-whitelysosymeantibodies. Ann. NY Acad. Sci. 418:140-50 42. Smith-Gill, S. J., Mainhart, C. R., Lavoie,T. B., Rudikoff,S., Potter, M. 1984. Vn-V L expression by monoclonal antibodiesrecognizingavian lysozyme. J. Immunol.132:963-67 43. Harper, M., Lema, F., Boulot, G., Poljak, R. J. 1987. Antigenspecificity and cross-reactivity of monoclonal anti-lysozymeantibodies. Mol. lmmunol. 2:97-108 44. Atassi, M.Z., Lee,C.-L. 1978.Theprecise and entire antigenic structure of native lysozyme.Biochem.J. 171: 42934 45. Mozes,E., Maron,E., Arnon,R., Sela, M. 1971. Strain-dependentdifferences in the specificity of antibodyresponses toward lysozyme. J. lmmunol. 106: 862-64 46. Prager, E. M., Wilson,A. C. 1971. The dependenceof immunologicalcrossreactivity uponsequenceresemblance among lysozymes. I. Micro-complementfixation studies. J. BioLChem. 246:5978~89 47. Amit,A. G., Mariuzza,R. A., Phillips, S. E. V., Poljak, R. J. 1985.Thethreedimensionalstructure of~ an antigenantibody complexat 6 A resolution. Nature 133:156-58 48. Amit,A. G., Mariuzza,R. A., Phillips, S. E. V., Poljak, R. J. 1986.Thethreedimensionalstructure of an antigenantibody complexat 2.8/~ resolution. Science 233:747-53 49. Blake, C. C. F., Koenig,D. F., Mair, G. A., North,A. C. T., Phillips, D.C., Sarma, V. R. 1965. Structure of hen egg-white lysozyme. A three-dimensional Fourier synthesis at 2/~ resolution. Nature206:757-61 50. Getzoff,E. D., Geysen,I-I. M., Rodda, S. J., Alexander,H., Tainer, J. A., Lerner, R. A. 1987. Mechanismsof antibodybinding to a protein. Science 235:1191-96 51. Chothia,C., Janin, J. 1975.Principles of protein-protein recognition. Nature 256:705~8 52. Honjo, T. 1983. Immunoglobulin genes. Ann. Rev. Immunol. 1: 499528 53. Tonegawa, S. 1983. Somaticgeneration of antibodydiversity. Nature302:57581 54. Chua, M. M., Goodgal,S. H., Karush, F. 1987. Germ-lineaffinity and germ-

577

line variable-region genes in the B cell response. J. Immunol.138: 128188 55. Colman, P. M., Laver, W. G., Varghese,J. N., Baker,A. T., Tulloch, P. A., Air, G. M., Webster,R. G. 1987. Three-dimensional structure of a complexof antibody with influenza virus neuraminidase.Nature326: 35863 56. Vargliese,J. N., Laver, W.G., Colman, P. M.1983. Structure of the influenza virus glycoprotein antigen neuraminidaseat 2.9 /~ resolution. Nature 303:35-40 57. Sheriff, S., Silverton, E. W., Padlan, E. A., Cohen,G. H., Smith-Gill, S., Finzel, B, C., Davies, D. R. 1987. Three-dimensional structure of an antibody/antigen complex. Proc. NatL Acad.Sci. USAIn press 58. Smith-Gill,S. J., Wilson,A. C., Potter, M., Prager, E. M., Feldmann,R. J., Mainhart, C. R. 1982. Mappingthe antigenic epitope for a monoclonal antibody against lysozyme..Llmmunol. 128:314-22 59. Guillon, V., Alzari, P. M., Poljak, R. J. 1987. Preliminary crystallographic study of a complexbetweenan heteroclitic anti-hen egg-whitelysozymeantibody and the heterologous antigen pheasant egg-whitelysozyme.J. Mol. BioL 197:375-76 60. De la Paz, P., Sutton, B. J., Darsley, M. J., Rees, A. R. 1986. Modelling of the combiningsites of three antilysozyme monoclonal antibodies and of the complexbetweenoneof the antibodies and its epitope. EMBO J. 5: 415-25 61. Roberts, S., Cheetham,J. C., Rees,A. R. 1987. Generation of an antibody with enhancedaffinity and specificity for its antigen by protein engineering. Nature 328:731-34 62. Jemmerson,R., Paterson, Y. 1986. Mappingepitopes on a protein antigen by the proteolysis of antigen-antibody complexes.Science 232:1001-4 63. Burnens,A., Demotz,S., Corradin,G., Binz, H., Bosshard, M. R. 1987. Epitope mapping by chemical modification of free antibody-bound protein antigen. Science 235:780-83 64. Barlow, D. J., Edwards, M. S., Thornton,J. M. 1986. Continuousand discontinuousprotein antigenic determinants. Nature 322:747-48 65. Atassi, M.Z. 1975.Antigenicstructure of myoglobin: the complete immunochemicalanatomyof a protein andconelusionsrelating to antigenicstructures

Annual Reviews

Annu. Rev. Immunol. 1988.6:555-580. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

578

ALZARI, LASCOMBE& POLJAK of proteins. Immunochemistry 12: 423Haber,E., Bruccoleri, R. E., Carlson, 38 W.E., Fanning, D. W., Smith, J. A., 66. Ibrahimi,I. M., Eder,J., Prager,E. M., Rose, G. D. 1986. Antigenic deterWilson, A. C., Arnon, R. 1980, The minantsin proteins coincide with sureffect of a single amino acid subface regions accessible to large probes stitution on the antigenicspecificity of (antibody domains).Proc. Natl. Acad. the loop region of lysozyme. Mol. Sci. USA83:226-30 Immunol. 17:37-46 79. Thornton, J. M., Edwards, M. S., 67. Sela, M. 1969. Antigenicity: some Taylor, W.R., Barlow, D. J. 1986. molecularaspects. Science 166: 1365Location of "continuous antigenic determinantsin the protrudingregions 69 of proteins. EMBO J. 5:409-13 68. Arnon, R., Maron, E., Sela, M., Anfinsen, C. B. 1971. Antibodies re80. Berzofsky, J. A. 1985. Intrinsic and active with native lysozymeelicited extrinsic factors in protein antigenic by a completely synthetic antigen. structure. Science 229:932~10 Proc. Natl. Acad. Sci. USA68: 145081. Tainer,J. A., Getzoff,E. D., Paterson, 55 A., Olson, A. J., Lerner, R. A. 1985. The atomic mobility component of 69. Langbeheim,H., Arnon, R., Sela, M, 1976. Antiviral effect on MS-2coliprotein antigenicity. Ann. Rev. Immuphage obtained with a synthetic nol. 3:423-38 antigen. Proc.Natl. Acad.Sci. USA73: 82. Moudallal,Z. AI., Briand, J. P., Van 4636-40 Regenmortel,M. H. V. 1985. A major 70. Audibert,F., Jolivet, M., Chedid,L., part of the polypeptide chain of Alouf,J. E., Boquet,P., Rivaille, P., tobaccomosaicvirus protein is antigenic. EMBO J. 4:1231-35 Siffert, O. 1981. Activeantitoxic immunizationby a diphtheria toxin syn83. Novotny,J., Haber, E. 1986. Static thetic oligopeptide. Nature289: 593accessibility modelof protein anti94 genicity: the case of scorpion neuro71. Lerner, R. A. 1982. Tappingthe immutoxin. Biochemistry25:6748-54 nological repertoire to produceanti84. Novotny, J., Handschumacher, M., bodies of predeterminedspecificity. Haber, E. 1986. Locationof antigenic Nature 299:592-96 epitopes antibody molecules. J. Mol. Biol.on189:715-21 72. Fraga, S. 1982.Theoretical prediction of protein antigenic determinantsfrom 85. Jemmerson,R. 1987. Chainflexibility amino acid sequences, Can. J. Chem. and antigenicity. Nature328:300 60:2606-10 86. Novotny, J., Bruccoleri,R. E., Carlson, 73. Hopp,T. P., Woods,K. R. 1981. PreW. D., Handschumacher,M., Haber, diction of protein antigenic deterE. 1987. Antigenicity of myohemeryminants from amino acid sequences. trin. Science.In press Proc. NatL Acad. Sci. USA78: 382487. Mariuzza,R, A., Phillips, S. E. V., 28 Poljak, R. J. 1987.Thestructural basis 74. Padlan,E. A. 1985.Quantitationof the of antigen-antibodyrecognition. Ann. immunogenic potential of protein antiRev. Biophys. Chem.16:139-59 gens. Mol. Immunol.22:1243-54 88. Oudin,J., Michel, M. 1963. Unenouvelle formed’allotypie des globulines 75. Westhof,E., Altschuh,D., Morns,D., Bloomer,A. C., Mondragon,A., Klug, gamma du strum de lapin appaA., VanRegenmortel,M. H. V. 1984. remment lib fi la fonction et la spbciCorrelation between segmental ficit6 des anticorps.C.R.Acad.Sci. 257: mobility and the location of antigenic 805-8 determinantsin proteins. Nature274: 89. Kunkel, H. G., Mannick, M., 92-94 Williams,R. C. 1963. Individual anti76. Tainer, J. A., Getzoff, E. D., genie specificities of isolated Alexander, H., Houghten, R. A., antibodies. Sc&nce140:1218-19 Olson,A. J., Lerner, R. A., Hendrick- 90. Davie, J, M., Seiden, M. V., Greenson, W.A. 1984.Thereactivity of antispan, N. S., Lutz, C. T., Bartholow,T. peptide antibodiesis a functionof the L., Clevinger, B. L. 1986. Structural atomicmobility of sites in a protein. correlates of idiotopes. Ann. Rev. Nature 312:127-34 Immunol. 4:147-65 77. Fanning, D. W., Smith, J. A., Rose, 91. Roux, K. H., Metzger, D. W. 1982. G. D. 1986. Molecularcartography of Immunoelectronmicroscopic localiglobular proteins with application to zation of idiotypes and allotypes on antigenic sites. Biopolymers25:863-83 immunoglobulinmolecules. J. Immu78. Novotny, J., Handschumacher, M,, nol. 129:780-83

Annual Reviews

Annu. Rev. Immunol. 1988.6:555-580. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

STRUCTURE OF ANTIBODIES 579 92. Roux,K. H., Monafo,W.J., Davie, J. M., Greenspan, N. S. 1987. Construction of an extendedthree-dimensional map by electron microscopic analysis of idiotope-anti-idiotopecomplexes. Proc.Natl. Acad.Sci. USA84: 4984-88 93. Boulot,G., Rojas, C., Bentley, G. A., Poljak, R. J., Barbier, E., Le Guern, C., Cazenave,P.-A. 1987. Preliminary crystallographic study of a complex betweenthe Fab fragment of a monoclonal anti-lysozymeantibody (D1.3) and the Fab fragment from an antiidiotypicantibodyagainsD1.3. J. Mol. Biol. 194:577~79 94. Oi, V. T., Morrison,S. L., Herzenberg, L. A., Berg, P. 1983. Immunoglobulin gene expression in transformed lymphoidcells. Proc.Natl. Acad.Sci. USA 80:825-29 95. Neuberger,M. S. 1983. Expressionand regulation of immunoglobulinheavy chain gene transfected into lymphoid cells. EMBO J. 2:1273-78 96. Ochi, A., Hawley,R. G., Hawley,T., Schulman, M. J., Traunecker, A., Kfhler, G., Hozumi,N. 1983. Functional immunoglobulinMproduction after transfection of cloned immunoglobulinheavyandlight chain genes into lymphoidcells. Proc. Natl. Acad. Sci. USA80:6351-55 97. Jones, P. T., Dear, P. H., Foote, J., Neuberger, M. S., Winter, G. 1986. Replacingthe complementarity-determining regions in a humanantibody with those from a mouse.Nature321: 522-25 98. Jencks, W.1969.Catalysis in Chemistry and Enzymolot?y. NewYork: McGraw Hill 99. Kohen,F., Kim,J. B., Lindner,H. R., Eshhar, Z., Green, B. 1980. Monoclonal immunoglobulin augments hydrolysis of an ester of the homologoushapten. Anesterase-like activity of the antibody-containingsite? FEBS Lett. 111: 427-31 100. Tramontano,A., Janda, K. D., Lerner, R. A. 1986. Catalytic antibodies. Science 234:1566-70 101. Pollack,S. J., Jacobs,J. W.,Schultz,P. G. 1986.Selectivechemicalcatalysis by an antibody. Science 234:1570-73 102. Jacobs,J., Schultz, P. G., Sugasawara, R., Powell, M. 1987. Catalytic antibodies. J. Am. Chem.Soc. 109: 217476 103. Menger, F. M., Ladika, M. 1987. Origin of rate accelerations in an enzymemodel:Thep-nitrophenyl ester syndrome. J. Am. Chem. Soc. 107:

3145-46 104. Kabat,E. A., Wu,T. T., Reid-Mitler, M., Perry, H. M., Gottesman, K. S. 1987. Sequencesof proteins of immunolot?ical interest. Tabulation and analysis of aminoacid andnucleic acid sequencesof precursors, V-regions,Cregions, J-chain, T-cell receptor for antigen,T-cell surfaceantigens,flz-microglobulins, majorhistocompatibility antigen, Thy-1, complement,C-reactive protein, thymopoietin, postgammaglobulin, and ~t2-macroglobulin. Washington, DC: US Dep. Health HumanServ. 4th ed. 105. Poljak, R. J., Amzel,L. M., Chen,B. C., Phizaekerly,R. P., Saul, F. 1974. Thethree-dimensionalstructure of the Fab fragment of a human myeloma immunoglobulin at 2.0 ,~ resolution. Proc. Natl. Acad. Sci. USA71: 3440106. Padlan, E. A., Davies, D. R., Pecht, I., Givol, D., Wright, C. 1977. Model building studies of antigen binding sites. The antigen binding site of MOPC315. Cold Sprint? HarborSyrup. Quant.Biol. 4:627-37 107. Dwek, R. A., Wain-Hobson, S., Dower,S., Gettings,P., Sutton,B., Perkins, S. J,, Givol,D. 1977.Structureof an antibody combining site by magnetic resonance. Nature266:31-37 108. Feldman, R. J., Potter, M., Glaudemans,C. P. J. 1981. A hypothetical space-filling modelof the V-regionsof the galactan-binding myelomaimmunoglobulin J539. MoLImmunoL 18:683-98 I09. Stanford, J. M,Wu,T. T. 1981.A predictive methodfor determining possible three-dimensional foldings of immunoglobulin backbones around antibody combiningsites. J. Theor. Biol. 88:421-39 110. Travers,P., Blundell,T. L., Sternberg, M.J. E., Bodmer,W.F. 1984. Structural and evolutionary analysis of HLA-D region products. Nature 310: 235-38 l 1 I. Novotny,J., Tonegawa, S., Saito, H., Kranz, D. M., Eisen, H. M. 1986. Secondary,tertiary and quaternarystructure of T-cell-specific immunoglobulinlike polypeptide chains. Proc. Natl. Acad. Sci. USA83:742-46 112. Chothia, C., Lesk, A. M. 1986. The relation between the divergence of sequence and structure in proteins. EMBOd. 5:823-26 113. Blundell, T. L., Sibanda, B. L., Sternberg, M. J. E., Thornton,J. M. .1987. Knowledge~based prediction of

Annual Reviews 580

ALZARI, LASCOMBE& POLJAK

Annu. Rev. Immunol. 1988.6:555-580. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

protein structures and the design of novel molecules. Nature 326: 34752 114. Snow,M. E., Amzel,L. M. 1986. Calculating three-dimensionalchangesin protein structure due to amino acid substitutions: the variable re#on of immunoglobulins.Proteins 1:267-79

115. Chothia, C., Lesk, A. M., Levitt, M., Amit,A. G., Mariuzza,R. A., Phillips, S. E. V., Poljak, R. J. 1986.Prediction of conformation of antigen-binding domains in immunoglobulins DI.3 andcomparisonwith crystal structure. Science 233:755-57

Annu. Rev. Immunol. 1988.6:555-580. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Annu. Rev. Immunol. 1988.6:555-580. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Annual Reviews www.annualreviews.org/aronline Ann.Re~. Immunol.1988. 6: 581-94

Annu. Rev. Immunol. 1988.6:581-594. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

PROSPECTS FOR GENE THERAPY FOR IMMUNODEFICIENCY 1DISEASES Phillip W. Kantoff,’~ Scott M. Freeman~f~and W. French Anderson~f ~" Laboratory of Molecular Hematology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland20892 :~ Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, Minnesota 55455

INTP~ODUCTION The fields of gene transfer and expression have recently seen major advances that have madepossible a better understanding of basic biologic mechanismsand have brought closer the possibility for gene therapy for the treatment of clinical disease. Vectors derived from murine retroviruses are being developedfor use in clinical protocols. In this article we address the recent advances in gene transfer as they relate to progress toward achieving gene therapy for immunodeficiencydiseases, specifically for adenosine deaminase (ADA)deficiency as a cause of severe combined immunodeficiency (SCID). It now appears that ADAdeficiency will the first disease in which the technique of retroviral-mediated gene therapy will be attempted.

METHODS OF GENE TRANSFER A number of techniques have been used successfully to introduce cloned genes into mammalian cells in culture (for a detailed review, see Ref. 1). The major methodscan be grouped in four categories: (a) viral, both RNA viruses (e.g. retroviruses) and DNAviruses (e.g. SV40, adenovirus, i The US Governmenthas the right to retain a nonexclusive royalty-free license in and to any copyright covering this paper.

581

Annual Reviews

Annu. Rev. Immunol. 1988.6:581-594. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

582

KANTOFF, FREEMAN & ANDERSON

bovine papilloma); (b) chemical, such as calcium phosphate-mediated DNA uptake; (c) physical, for example, microinjection and electroporation; and (d) fusion, that is, fusion to target cells of DNA-loaded membranous vesicles, such as liposomes, red blood cell ghosts, or protoplasts. Retrovirus-basedvectors have becomethe technique of choice for many in vitro andin vivo genetransfer experiments;these are discussedbelow. Several DNA viruses have been engineered to be used as vectors for the transfection of genes in vitro. Considerablesuccess has been obtained using DNA virus-based vectors in the transfer of selectable markergenes and, in certain circumstances,nonselectablegenesinto tissue culture cells. However,studies using these vectors in situations wherehigh efficiency stable integration is required, such as with bonemarrowstemcells, have not yet been reported. Themainchemicalmethods,calciumphosphateand DEAE dextran, continue to be a mainstayin molecularbiology becauseof their ease of performance; however,they are limited by their relatively low efficiency (typically less than 1:10 3 cells transduced).Similarly, fusion techniques and electroporation (the latter uses an electric current to mediate DNA uptake into cells) are effective on transformed cells but appear to produce a lower level of gene transfer in primary(nontransformed)cells. In recent years microinjection of genes into fertilized mouseova with subsequent implantation of the egg(s) into pseudopregnantanimals order to producetransgenic offspring has providedan elegant meansfor examiningtissue-specific geneactivity. Theneedfor specialized equipment and trained personnel has limited the widespreaduse of this technique. Furthermore,genetransfer into humanova for treatment of genetic disease (which wouldbe germline gene therapy) is not currently an acceptable approach.Nevertheless, microinjection will continue to provide a means for answeringquestions about geneactivity in in vivo animalsystems. Theengineeringof retroviruses as vectors for genetransfer has expanded greatly in recent years and offers at the current time the mostpromisefor clinical use in genetherapy(1). USE OF RETROVIRAL

VECTORS

A numberof features of the retrovirus makethese agents particularly useful as a delivery system for genes. Part of the retrovirus life cycle involvesthe stable integration of its genetic material into the host genome (in its DNA form, called a provirus). A detailed review of the structure and life cycle of the retrovirus has been published(2), as have reviews describing howretroviral vectors are constructed and used (3-6). Only

Annual Reviews

Annu. Rev. Immunol. 1988.6:581-594. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

PROSPECTS FOR GENE THERAPY

583

those features relevant to the construction and use of retroviral vectors (primarily those based on Moloneymurine leukemia virus) are highlighted here. A retrovirus is composed of a glycoprotein envelope and an RNAprotein core. It is the envelopethat recognizes a cell surface receptor and determines host specificity. After the virus binds to the cell surface, the RNA-protein core (composed of the viral RNA,structural proteins, and a reverse transcriptase) enters the cell where the viral RNAis reverse transcribed into linear DNA.The linear double stranded DNAis transported to the nucleus where it is ligated to form closed circular DNAand is then integrated into the host cell’s genome. The site of integration appears to be selected randomly in the cellular genome although some evidence suggests that it might occur more often at transcriptionally active sites. Onceintegrated, the provirus is transcribed into viral messenger RNA,some of which is used for viral protein production. Viral proteins and viral RNAcombine to form viral cores which bud through regions of the cellular membranethat have already incorporated viral envelope proteins. These membrane-wrapped cores are released from the cell as new viral (type C) particles. In creating a retroviral vector there are several considerations to be met: (a) The vector should be compatible with the production of a large number of viral particles (i.e. a high titer) in permissivecells so that there can efficient transfer of the vector’s gene(s) to target cells; (b) the vector should permit the expression of the transmitted gene(s) in the target cells; and (c) the vector should be stable in the target cells. The functions of a retrovirus can be divided into two types whenconsidering the construction of retroviral vectors: trans and cis. Structural features required in trans (i.e. supplying the core proteins, core enzymes, and envelope glycoprotein) can be provided separately whereas functions required only in cis (e.g. sequences necessary for genomeintegration, gene expression and packaging of the genomic RNA)need to be built into the vector. Trans functions can be supplied by using so-called "packaging cell lines" (7-10) which can provide all the trans functions necessary for virus production. These cell lines produce emptyvirus particles (i.e. particles containing no viral RNA)and are made as follows. A defective helper virus is constructed that carries all the viral genes (gag, pol, env) but has had the packaging signal (the cis function necessary for the genomicRNA to enter the viral particle) deleted. This virus is transfected into a permissive cell line, e.g. NIH3T3cells; the result is a cell that can makeviral particles which contain no RNA.Whena retroviral vector (containing a packaging sequence, a gene(s) of choice, but no viral genes) is introduced into this "packaging cell line," the vector RNAis encapsidated into the previously

Annual Reviews

Annu. Rev. Immunol. 1988.6:581-594. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

584

KANTOFF, FREEMAN & ANDERSON

defective viral particles. Virions are thus producedthat are capable of carrying out one further roundof infection andintegration of viral genetic material (becausethey haveall the viral proteins necessary)but that not havethe ability to makeany moreviruses becauseno viral genesare present. Theminimalstructural features of a retroviral vector required in cis are both LTRs,the primer binding sites, and the packagingsignal. Althougha great deal has beenlearnedaboutthese sequences,there is still considerable uncertaintyabout manydetails. Asa result, retroviral-mediatedgenetransfer is still hampered by the variability of expressionof transducedgenes in vivo. It appears that, for unknownreasons, somevectors are more suitable for the in vivo expressionof genesthan are others. Anexampleof a very successful retroviral vector is N2(Figure 1). All that remains of the original (8.3 kb) Moloneymurineleukemicprovirus are: (a) the 5’ LTRwith the associated primerbindingregion, the 5’ donor site (labeled 5’), the packagingsequence(labeled $), andseveral hundred basesincluding418bases of the gagprotein (total, 1.5 kb), and (b) the LTRwith its associatedbinding site and a short stretch of viral sequences (total, 0.8 kb). In place of the 6.0 kb removedis 1.5 kb of bacterial DNA that contains a neomycinresistance gene(crosshatchedarea in Figure 1). TheN2vector has been successfully used in a numberof in vitro and in vivo experiments(11-14). A detailed description of the constructionof the N2vector has been published(15). POTENTIAL DEFICIENCY

FOR GENE THERAPY

IN

ADA

Natural History of ADA Deficiency Severe combinedimmunedeficiency (SCID)is the most profound of the primaryimmune deficiency diseases. It is characterized by a dual system immune defect with virtual absenceof all T and B cell immunefunction. Byits nature, SCIDis a disease of infancy; mostaffected individuals die of infection by two years of age. In about onefourth of all SCIDcases, a deficiency of the purine catabolic enzymeadenosinedeaminase(ADA)has been found. A numberof excellent reviews (16--18) as well as a recent symposium (19) have discussed ADA deficiency in detail. SCIDpatients are generally agammaglobulinemic and fail to produce antibodies following immunizationor infection. They also generally exhibit striking lymphopeniaand are unable to mountT cell immune responses.This thereby makesthemsusceptible to serious or fatal infections with opportunistic organisms.Theseinfants have a clinical course featuring recurrent infections with all classes of microorganisms involvingthe

Annual Reviews PROSPECTS FOR GENE THERAPY S

LTR

0.5

P P

E

P

P XEC

R NEO

5’ ¯

1.0

1.5

2.0

2.5

585

S

LTR

3.0

3.5

4.0

Annu. Rev. Immunol. 1988.6:581-594. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

SCALE IN Kb FigureI Diagramof the integrated vector (proviral) N2.0to 1.5 and3.0 to 3.8 kb: Moloney murineleukemiavirus sequence;1.5 to 3.0 kb box: Tn5sequencecontaining the neoR gene (Bgl I-BamHI fragment from Tn5); the hatched area is the coding sequence. LTR,long terminalrepeat; 5’, the donorsplice site at the 5’ end; ~, packagingsequence;restriction enzyme sites: S, SacI; P, Pst I; E, EcoRI; X, XhoI; C, Cla I.

skin, central nervoussystem, sinopulmonary and gastrointestinal tracts. Theaffected children usually fail to thrive and havechronicdiarrhea and malabsorption,skin rashes, and a high incidence oflymphoma. Thedisease is uniformly lethal. SCIDcan occur in both autosomal recessive and X-linked forms; the biochemical basis for most cases is unknown. ADA(-)SCID is inherited as an autosomal recessive. The heterozygote parents are diagnosable because they have approximately 50%of the normalconcentrationof ADA in their cells. Adenosinedeaminasecatalyzes the conversion of adenosine (Ado)and deoxyadenosine (dAdo)to inosine and deoxyinosine, respectively. In the absence of ADA,dAdoaccumulates intracellularly (as well as extracellularly) and becomesphosphorylated to dATP,a compoundnormally not detected at high levels in mammalian cells. Althoughthe exact mechanismis still unclear, it is known that severeimmune deficiencyis a direct consequence of the deficiency of ADA.The production of dATPfrom dAdois involved in the cellular toxicity observed. AlthoughADAis deficient in all the cells of the body,T cells havethe highestconcentrations of any cell in the bodyof the enzymeswhichphosphorylatedAdoto dATP. This probablyaccounts for the observation that immunodeficiency is the prominent pathological consequenceof ADA deficiency (20). Furthermore, whendeoxycoformycin (a potent inhibitor of ADA)is used in the treatment of acute lymphocytic leukemia, a similar degree of immune deficiencyis seen to developin the patient (21, 22). The immunologic abnormalities commonin ADA(-)SCIDinclude striking depletion of T lymphocytesin all lymphoidorgans. Thepatients are anergic, andmanypatients haveno detectableT cells in their peripheral blood, while a few mayhave up to 30-40%of normal T cell counts.

Annual Reviews

Annu. Rev. Immunol. 1988.6:581-594. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

586

KANTOFF,

FREEMAN & ANDERSON

Despite the presence of T cells in the periphery, in vitro assays of T cell functions showseverely defective responses. Thepatient’s cells may proliferate to a limited extent in response to strong mitogens(such as PHA),but the cells showno response to soluble antigens such as tetanus toxoidor to allogeneiccells in in vitro assays. Immune responsesin patients as well as peripheral T cell numbersmayvary in patients over time. B lymphocytenumbersvary considerably morethan do T cells in these patients. Although all patients have hypogammaglobulinemia, B cell counts can range from very low to elevated. Nonetheless,patients cannot mountan immuneresponse to immunizations.In in vitro studies, these B cells can sometimesbe inducedto secrete immunoglobulins if T cells from normaldonors are addedto the cultures (23). This observation suggests that the dysfunctionof B cells is not dueto anyintrinsic defect but rather to a lack of normalT cell help. ADA(-)SCID is a goodcandidate disease for gene therapy for several reasons. It is invariably fatal if untreated. Treatmentof ADA deficiency by transplantation with bone marrowfrom an HLA-identicalsibling donor results in the correction of the defective immune systemin these patients; however,only one third of patients have a matcheddonor. Further, ADA deficiency has no major nonimmunologic features that need correction or whichcannot be corrected by bonemarrowtransplantation (24-27). Thus, successful insertion of a functioning ADA gene into the patient’s own bonemarrowcells shouldbe sufficient to correct all clinically significant manifestationsof the disease. Theresults of bonemarrowtransplantation also suggest that correction of the enzymedefect in the patient’s T lymphocytes alone maybe sufficient to result in full immunologicreconstitution. Several ADA(-)SCID patients whoseimmunesystems have been fully reconstituted by meansof matched bone marrowtransplantation have T cells as the only donor type cell remainingbeyondthe immediate post-transplant period (27). Furthermore,corrected cells should have selective growthadvantage in the patient, and consequently, no cytoablation of the patient’s ownmarrowwouldbe necessary. Avery wide range of ADA levels has been observedin different individuals whohave normal immunefunction (28). Heterozygotes for ADA deficiency are immunologicallynormaldespite their having only 50%of the usual ADA levels. Rareindividuals (e.g. the African !Kungtribesmen and several children found by screening in NewYork State) have been studied and found to have only 5-10%of normal ADA,and yet they are also immunologicallynormal. Thus, it maynot be necessary to achieve completely normal ADAlevels by gene therapy to have a beneficial outcome.Similarly, a few individuals havebeendescribed with ADA levels 40-50 times higher than normal in their erythrocytes (although normal

Annual Reviews PROSPECTS FOR GENE THERAPY

587

Annu. Rev. Immunol. 1988.6:581-594. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

levels in other cells) (29). These patients have a mild form of hemolytic anemia but are also immunologically normal. Therefore, expression of the transferred ADAgene severalfold above the normal level may also be acceptable clinically. This very broad range of ADAgene expression in healthy individuals suggests that a functioning ADAgene inserted via retroviral-mediated gene transfer need not be closely regulated for the procedure to benefit the patient. Finally, the gene for ADAhas been cloned and is available as a cDNA(30-32).

In Vitro Studies Using Retroviral Vectors Containing the Human ADA cDNA The initial experiments in this field centered around the construction of retroviral vectors that would transmit a functioning ADAgene into cultured cells. Valerio et al (33) and Friedman (34) independently inserted the human ADAcDNAinto the retroviral vector pZIP-NeoSV(X) that had been constructed by Cepkoet al (35) (Figure 2). Valerio et al found that their recombinantvirus infected NIH3T3 cells and expressed an equal amount of human and endogenous mouse ADA. Similarly, Friedman demonstrated that the recombinant virus could be used to transduce a ADA-CONTAINING VECTORS pZIP-NeoSV(X)~~.~.’. ADA

~

9~:~

S

ori

P

ori

[~

SAX

E ~ ~ ~ ~ ] ~" ~A~A." .~." .~://////~1~ Figure 2 Schematic diagrams of the various retrovirus vectors containing the humancDNA for adenosine deaminase(ADA).See text for a description of each vector and the reference(s) for the relevant publication. Abbreviations: LTR,long terminal repeat; D, 5’ donor splice site; ~b, packaging sequence; ADA,adenosine deaminase; A, 3’ receptor splice site; Neo, neomycinresistance gene; S ori, origin for SV40;P ori, origin from polyomavirus; S, SV40 early gene promoter; DHFR,dihydrofolate reductase; HPRT,hypoguanine phosphoribosyl transferase; M, metallothionein promoter; P, phosphoglycerokinase promoter.

Annual Reviews

Annu. Rev. Immunol. 1988.6:581-594. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

588

KANTOFF,

FREEMAN & ANDERSON

mouseT cell lymphoma line BL/VL3.In the infected cells humanactivity was present at 25%-50% of the endogenousmouseADAactivity. Kantoff et al (36) inserted an SV40-promotedhumanADAcDNAinto the vector, creating a vector called SAX.This vector wasused to infect ADAdeficient humanT (TJF-2) and B (GM2756 and GM4258A) cell lines that had been immortalized with HTLV-Iand EBV,respectively. They found that humanADAwas produced in these cells in amounts comparable to those foundin normalT and B cells. This level of activity was sufficient to correct the altered sensitivity of TJF-2to 2’ deoxyadenosine characteristic of the ADA-deficient state. Usinga vector called pZIP-NeoSV(B)(35), Belmontet al (37) inserted human ADAcDNA, creating the vector SVBADA211. They infected murine bone marrowcells and demonstratedexpression of the neo R gene in approximately 10%of the cells by colony formation (CFU-C)in the presence of G418.Marrowcells whichhad been infected and selected in G418were pooled to successfully demonstrate the production of human ADAexpression using a mediumcontaining a combination of 9-xylofuranosyl adenine (XylA) and deoxycoformycin,a potent inhibitor ADA. This paper demonstratedthe utility of metabolicselection of cells for ADA to improvegene transfer efficiency. It also wasthe first demonstration of ADA activity after gene transfer in nonimmortalized primary cells. Palmeret al (38), using a vector similar to SAX,demonstratedthat humanADA-deficientdiploid skin fibroblasts could be infected and would then express humanADA.Similarly, Kohnet al (39) using the SAXvector demonstrated that ADA-deficient peripheral T lymphocytes could be infected and would then express humanADA. In Vivo Studies Using Retroviral Vectors Containing the ADA Gene Despite the generally encouragingresults of ADA expression obtained in the in vi.tro studies, the in vivo studies in animalmodelsreported thus far havebeendisappointing.Williamset al (40) used vectors basedon a parent structure called pZIP-SV(B),except that the neoR gene in pZIP-SV(B) vectors had been replaced by a mutant dihydrofolate reductase gene (DHFR*).Inserted into this vector was humanADA cDNA,either alone or promotedby the SV40early promoter. Althoughthe vector with just the ADAcDNAfunctioned poorly, the vector with the SV40promoter (ZIPDHR* SVADA, Figure 2) permitted efficient expression of human ADAin a murine pre-B cell line (70Z), a murine thymomacell line (BW5127),and an ADA-deficientB cell line (GM2471). Infection murinebone marrowtransplanted into lethally irradiated syngeneichosts

Annual Reviews

Annu. Rev. Immunol. 1988.6:581-594. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

PROSPECTS

FOR GENE THERAPY

589

demonstrated efficient transfer of the vector into spleenhematopoieticcells 2 weeks after transplant. However,no expression of the humanADA protein was detected. WhenRNA analysis was performedby meansof S 1 nuclease assay, low levels of transcripts initiating in the 5’LTRwere detected, but no RNAinitiated from the SV40promoterwasseen. Similar results with regard to the absence of ADAprotein production were obtained by Zwiebelet al (41) using the SV40-promoted ADA vector SAX whichhad been used successfully to infect and express ADA in humanT cells. Microinjection studies using an SV40-promoted DHFR gene (42) or an SV40-promoted T antigen (43) have shownpoor tissue-specific expressionof these genesin hematopoieticcells of the mouse.This suggests that the SV40promotermaybe an inappropriate choice for vectors used in murinebone marrowgene transfer. Thebacterial neoR genecodesfor a phosphotransferasethat inactivates neomycin-likeantibiotics. G418,a neomycinanaloguethat is lethal to mammalian cells, can be used for positive selection of cells carrying the neoR gene. Magliet al (44) demonstrated that a retroviral vector containing an SV40-promotedneo~ gene when used to infect bone marrowcells formedsmaller G418-resistant CFU-Cin vitro, and no G418-resistant CFU-C derived from progenyspleen colonies. In contrast, a vector containing a herpes simplex virus (HSV)thymidine kinase (TK)-promoted neo~ gene formed large G418-resistant CFU-Cin vitro and abundant G418-resistant CFU-Cderived from spleen colonies. Thesedata suggest, again, that there maybe poor transcription from the SV40promoter in murinehematopoieticcells. Before concluding, however,that the HSVTK promotermightbe appropriate for murinehematopoieticcells, it should be noted that Zwiebelet al (41) haveshownthe absenceof expression murineCFU-Susing a vector similar to SAXin whichthe SV40promoter has been replaced by the HSVTK promoter. Thus, although the choice of a promoter maybe importanL the vector construction must also be permissive. In a similar study, Mclvoret al (45) saw no ADA activity in mouse spleen foci using vectors with HPRT as the dominantselectable marker and hADApromoted by the mouse metallothionein promoter (LHMS AL, Figure 2). Encouragingresults were recently obtained by Limet al (46) using a vector containing a phosphoglycerokinase-promoted human ADAcDNA(PGK-ADA)and by P. W. Kantoff, M. A. Eglitis and W. F. Anderson(unpublished observation) using a vector derived from N2, called pB2A.In both cases humanADA was detected in a small percentage of murine spleen colonies after infection of bone marrowand transplantation into lethally irradiated hosts. Thetiters of the vectors fromboth groupsappear to be too low to study expressionin fully recon-

Annual Reviews 590

KANTOFF, FREEMAN & ANDERSON

stituted animals.Notably,in both cases, the vectorslackedselectable genes colinear with the ADA gene.

Annu. Rev. Immunol. 1988.6:581-594. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

ADA Gene Transfer

Into

Monkeys

A monkeyautologous bone marrowtransplantation/~ene transfer protocol (using both Cynomolgusmacaqueand rhesus) was developed Kantoff et al (47). The protocol is outlined in Figure 3. Bonemarrow cells are removedfrom a youngadult monkey,the mononuclearcells are isolated by Ficoll centrifugation, andthe cells are then incubatedwith the retroviral vector SAXfor 2 hr at 37°C. Afterwardsthe cells are washed, resuspendedin autologousserum, and reinjected IV into the sameanimal. Just prior to receiving the marrowcells, the animalis subjectedto lethal irradiation (1000rad). Since there is no animalmodelfor ADA deficiency, PRIMATEAUTOLOGOUS B MT/GENE TRANSFER PROTOCOL bonemarrow cells

Infect BMcells withretroviralvector

~BM ~lls

~

~~

~ Viral

pa~es

Infuse >1! BMcells backinto irradiated monkey

~

>3 weeks

Reconstitution

Obtainperipheralbloodand BMfor biochemical analysis Figure 3 Protocol for gene transfer via autologous bone marrow transplantation into monkeys. See text and reference 47 for details. Abbreviations: BMT,bone marrowtransplantation; BM, bone marrow.

Annual Reviews

Annu. Rev. Immunol. 1988.6:581-594. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

PROSPECTS FOR GENE THERAPY

591

the monkeysrequired lethal irradiation in order to ablate their remaining marrowand to makespace for the treated cells. Following transplantation, the animals received blood products, antibiotics, and nutritional support by means of an indwelling Broviac catheter until they achieved normal hematopoietic function and reconstitution of all blood lines. At intervals after transplantation, blood and bone marrowsamples were analyzed for vector DNAsequences by Southern blots, and for both human ADAand neoR phosphotransferase enzymeactivities. The authors demonstrated low (up to 0.5% of endogenous primate ADAactivity) but readily detectable levels of humanADAin peripheral blood and bone marrowfrom several primates after gene transfer, as well as neoR phosphotransferase activity. Despite the achieved expression of humanADAin the primates, the levels of hADAactivity diminished with time and disappeared 4-5 months after transplant. There was, however, detectable evidence of persistent vector activity (G418 resistence) in lymphocytes up to 256 days after transplant. The authors concluded that the main obstacle to obtaining higher and more persistent levels of hADA in the primates was the poor infectivity of primate bone marrowstem cells by the amphotropic-enveloped murine retroviral vector. Expression of humanADAfrom the vector appeared to be substantial in those few cells (0.5%) which contained the vector DNA.

FUTURE APPLICATIONS OF GENE THERAPY FOR IMMUNODEFICIENCY DISEASE The ability to transfer genes into bone marrow stem cells of mice and monkeyswith in vivo expression of that gene has now been achieved. The human ADAgene can be transferred and expressed in primate bone marrow, thus bringing humanapplication ofgene transfer one step closer. Although the ability to transfer the ADAgene to primate bone marrow in vivo is relatively inefficient (0.5%), this level maybe adequate to cure ADAdeficiency. These patients are immunodeficient due to an inability to metabolize deoxyadenosine which accumulates and results in toxicity to the T cells. Therefore, T cells expressing the ADAgene should have a selective advantage in vivo, and even a small percentage of ADA-expressing bone marrowprogenitor cells may permit T lymphocyte reconstitution of the patient. Although the number of ADAdeficiency patients is very small, this disease will probably serve as a modelsystem for other applications of the new therapeutic regimen of gene therapy. Other immunodeficiencydiseases are candidates, but first the gene causing the disease must be identified and cloned. At present, only the genes for purine nucleoside phosphorylase

Annual Reviews

592

KANTOFF, FREEMAN& ANDERSON

Annu. Rev. Immunol. 1988.6:581-594. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

(PNP)deficiency andchronic granulomatous disease havebeen cloned, and the PNPgene has been placed in a retroviral vector and shownto express the enzymein vitro. Otherimmunodeficiency disease that are potential candidatesfor gene therapyin the future are: Chediak-Higashi syndrome,LFA-1deficiency, ,reticular dysgenesis, Swiss-typeagammaglobulinemia, X-linked agammaglobulinemia, Brutons type agammaglobulinemia,and bare lymphocytesyndrome.

Literature Cited 1. Anderson, W. F. 1984. Prospects for humangene therapy. Science 226: 4019 2. Varmus, H., Swanstrom, R. 1982. In RNATumorViruses, ed. R. Weiss, N. Teich, H. Varmus,J. Coffin, pp. 369512. Cold Spring Harbor, NY: Cold Spring Harbor Lab. 3. Temin,H. M. 1986. Retrovirus vectors for genetransfer. In GeneTransfer, ed. R. Kucherlapata, pp. 149-87. New York: Plenum 4. Mulligan, R. C. 1983. Construction of highly transmissible mammalian cloning vehicles derived from murine retroviruses. In ExperimentalManipulation of Gene Expression, ed. M. Inouye, pp. 155-78. NewYo~k:Academic 5. Dick, J. E., Magli, M.C., Phillips, R. A., Bernstein, A. 1986.Geneticmanipulation of hematopoieticstem cells with retrovirus vectors. TrendsGenet.2(6): 165-70 6. Gilboa, E., Eglitis, M.A., Kantoff, P. W., Anderson,W.F. 1986. Transfer and expressionof clonedgenes using retroviral vectors. BioTechniques4:504-12 7. Mann,R., Mulligan, R. C., Baltimore, D. 1983. Construction of a retrovirus packagingmutantand its use to produce helper free defectiveretrovirus. Cell 33: 153-59 8. Miller, A. D., Law,M.F., Verma,I. M. 1985. Generationof helper-free amphotropic retroviruses that transduce a dominant-acting, methotrexate-resistant dihydrofolate reductase gene. Mol. Cell Biol. 5:431-37 9. Sorge, J., Wright, D., Erdman,V. D., Cutting, A. E. 1984. Amphotropic retrovirus vector systemfor humancell gene transfer. MoLCell Biol. 4:1730-37 10. Miller, A. D., Buttimore,C. 1986. Redesignof retrovirus packagingcell lines to avoid recombination leading to helper virus production. MoLCell BioL 6:2895-2902

ll. Keller, G., Paige, C., Gilboa, E., Wagner, E. F. 1985.Expressionof a foreign gene in myeloid and lymphoid cells derived from multipotent haematopoietic precursors. Nature14:149-54 12. Dick, J. E., Magli, M. C., Huszar, D., Phillips, R. A., Bernstein,A.1985.Introductionof a selectable geneinto primitive stem cells capable of long-term reconstitution of the hemopoieticsystem v mice. Cell 42:71-79 ofWW 13. Eglitis, M. A., Kantoff, P., Gilboa,E., Anderson,W.F. 1985. Geneexpression in miceafter high efficiencyretroviralmediated gene transfer. Science 230: 1395-98 14. Hogge,D. E., Humphries,R. K. 1987. Genetransfer to primary normal and malignant human hemopoietic progenitors using recombinantretroviruses. Blood 69:611-17 15. Armentano,D., Yu. S.-F., Kantoff, P. W., von Ruden, T., Anderson, W. F., Gilboa,E. 1987.Effect of internal viral sequenceson the utility ofretroviral vectors. d. Virol. 61:1647-50 L. F., Seegmiller,J. E. 1980. 16. Thompson, Adenosine deaminase deficiency and SCID.Adv. Enzymol. 51:167-210 17. Martin,D. W.Jr., Gelfand,E. W.1981. Biochemistry of diseases of immunodevelopment. Ann. Rev. Biochem.50: 845-77 18. Kredich,N. M., Hershfield, M.A. 1983. Immunodeficiencydiseases caused by adenosinedeaminasedeficiency and purine nucleosidephosphorylasedeficiency. In The Metabolic Basis of Inherited Disease, ed. J. B. Stanbury,J. D. Wyngaarden, D. S. Fredrickson, J. L. Goldstein, M. S. Brown. NewYork: McGraw-Hill.5th ed. 19. Tritsch, G. L., ed. 1985. Adenosine Deaminase in Disordersof PurineMetabolism and in ImmuneDeficiency. Annals N.Y. Acad.Sci. Vol. 451 20. Seegmiller,J. E. 1985.Overviewofposs-

Annual Reviews

Annu. Rev. Immunol. 1988.6:581-594. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

PROSPECTS FOR GENE THERAPY

593

ible relation of defects in purinemetabence 195:783-85 olism to immune deficiency. Ann. N.Y. 30. Wiginton,D. A., Adrian, G. S., FriedAcad.Sci. 451:9-19 man,R. L., Suttle, D. P., Hutton,J. J. 21. Grever, M. R., Slaw, M. F. E., Jacob, 1983. Cloning of eDNAsequences of W.F., Aneidhart, J. A., Miser, J. S., human adenosine deaminase. Proc. Coleman,M. S., Hutton, J. J., BalcerNatl. Acad. Sci. USA80:7481-85 zak, S. P. 1981. The biochemical and 31. Valerio, D., Mclvor,R. S., Williams, clinical consequenceof 2’-deoxycoS. R., Duyvesteyn, M. G. C., van formycinin refractory lymphoproliferOrmondt,H., van der Eb, A. J., Martin, ative malignancy.Blood 57:406-17 D. W. 1984. Cloning of humanaden22. Hershfield,M., Kredich,N., Falletta, J., osine deaminasecDNAand expression Kinney,T., Mitchell,B., Koller,C. 1981. in mousecells. Gene31:147-53 An in vivo model of adenosine de32. Daddona,P. E., Shewach,D. S., Kelley, aminase (ADA)deficiency. Clin. Res. W. N., Argos, P., Markhan, A. F., 29:513 Orkin, S. H. 1984. Humanadenosine 23. Buckley,R. H., Gilbertsen,R. B., Schiff, deaminase cDNAand complete primary R. I., Ferreira, E., Sanal, S. O., Waldaminoacid sequence.J. Biol. Chem.259: mann, T. A. 1976. Heterogeneity of 12101-6 lymphocytesubpopulations in severe 33. Valerio, D., Duyvesteyn, M. G. C., combinedimmunodeficiency.Evidence van der Eb, A. J. 1984. Introduction of againsta stemcell defect.J. Clin. Invest. sequences encoding functional human 58:130-36 adenosine deaminaseinto mousecells 24. Parkman,R., Gelfand,E. W., Rosen,F. using a retroviral shuttle system. Gene 34:163q58 S., Sanderson,A., Hirschhorn,R. 1975. Severe combined immunodeficiency 34. Friedman, R. L. 1985. Expression of and adenosinedeaminasedeficiency. N. humanadenosine deaminase using a Engl. J. Med.292:714-19 transmissible murineretrovirus vector 25. Chert, H. S., Ochs, H. P., Scott, C. system. Proc. Natl. Acad.Sci. USA82: R., Gilblett, E. R., Tingle, A. J. 1978. 703-7 Adenosinedeaminasedeficiency: Dis- 35. Cepko,C. L., Roberts, B. E., Mulligan, appearance of adenosine deoxynucleoR. C. 1984. Construction and applitides froma patient’s erythrocytesafter cations of a highlytransmissible murine successful bone marrowtransplantaretrovirus shuttle vector. Cell 37: 1053tion. J. Clin. Invest. 62:1368-89 62 26. Hirschhorn, R., Roegner-Maniscalco, 36. Kantoff, P. W., Kohn,D. B., Mitsuya, V., Kuristsky, L., Rosen,F. S. 1981. H., Armentano,D., Sieberg, M., ZwieBonemarrowtransplantation only parbel, J. A., Eglitis, M., McLachlin, J. R., tially restorespurinemetabolitesto norWiginton,D. A., Hutton, J. J., Horomal in adenosine deaminasedeficiency witz, S. D., Gilboa, E., Blaese, R. M., patients. J. Clin. Invest. 68:1387-93 Anderson, W.F. 1986. Correction of 27. O’Reilly,R. J., Kapoor,N., Pollack, M. adenosinedeaminasedeficiency in culS., Chaganti,R. S. K., Dupont,B., Kirktured humanT and B cells by retroviruspatrick, D., Reisner, Y. 1982. Immunomediated gene transfer. Proc. Natl. logic function in patients transplanted Acad. Sci. USA83:6563-67 for severe combinedimmunodeficieney 37. Belmont, J. W., Henkel-Tigges, J., selectively engrafted with donor T Chang,S. M.W., Wager-Smith, K., Kellymphocytes. International Workshop: lems, R. E., Kick, J. E., Magli,M.C., The influence of the thymus on the Phillips, R. A., Bernstein,A., Caskey,C. generation of the T-cell repertoire. T. 1986. Expressionof humanadenosine Rudesheim, West Germany. Sept. deaminase in murine haematopoietic 1981.Behrint?Institute Mitteilun#en70: progenitor cells following retroviral 182-87 transfer. Nature322:385-87 28. Hirschhorn,R. 1985. Completeand par- 38. Palmer, T. D., Hock, R. A., Osborne, tial adenosine deaminasedeficiency: W.R. A., Miller, A. D. 1987. Efficient Relationship of immunefunction to retrovirus-mediatedtransfer andexpresmetabolite concentrations, enzyme sion of a human adenosine deaminactivity, and effects of therapy. Ann. ase gene in diploid skin fibroblasts N.Y. Acad. Sci. 451:20-25 from an adenosinedeaminase-deficient 29. Valentine, W.N., Paglia, D. E., Tarhuman.Proc. Natl. Acad. Sci. USA84: taglia, P., Gilsanz,F. 1977.Hereditary 1055-59 hemolyticanemiawith increased aden- 39. Kohn,D., Zwiebel,J., Blaese, R. M., osine deaminase (45-70 fold) and Anderson, W. F. 1988. Transfer and decreasedadenosinetriphosphate. Sciexpression of the ADAgene in ADA

Annual Reviews

Annu. Rev. Immunol. 1988.6:581-594. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

594

KANTOFF, FREEMAN& ANDERSON

deficient humanlymphocytes using tiple hematopoieticcell lineages folretroviral vectors. Submitted lowingretroviral vector gene transfer. 40. Williams,D. A., Orkin, S. H., Mulligan, Proc. Natl. Acad.Sci. USA84:789-93 R. C. 1986oRetrovirus-mediatedtrans- 45. Mclvor,R. S., Johnson,M. J., Miller, fer of humanadenosinedeaminasegene A. D., Pitts, S., Williams,S. R., Valerio, sequencesinto cells in culture and into D., Martin, D. W. Jr., Verma,I. M. 1987. Humanpurine nucleoside phosmudnehematopoieticcells in vivo. Proc. Natl. Acad. Sci. USA83:2566-70 phorylase and adenosine deaminase: 41. Zwiebel,J. A., Kantoff,P. W., Eglitis, gene transfer into cultured cells and M. A., Kohn, D., Muenchau, D., murine hematopoietic stem cells by McLachlin,J. R., Karson, E., Wieder, using recombinantamphotropicretroR., Yu,S. F., Blaese, R. M., Gilboa,E., viruses. Mol.Cell. Biol. 7(2): 838-46 46. Lim,B., Williams,D. A., Orkin, S. H. Anderson, W. F. 1986. Genetransfer and expressionusing a family of retro1987. Retrovirus-mediatedgene transviral vectors. Blood68: 307a fer of humanadenosine deaminase:Ex42. Isola, L. M., Gordon,J. W.1986. Syspression of functional enzymein murine hematopoieticstem cells in vivo. Mol. temic resistance to methotrexate in transgenic mice carrying a mutant Cell. Biol. 7(10): 3459-65 DHFRgene. PNAS 83:9621-25 47. Kantoff, P. W., Gillio, A., McLachlin, T. A., Finlay, C., Miller, D., J. R., Bordignon,C., Eglitis, M. A., 43. VanDyke, Marks,J., Lozno,G., Levine, A. 1987. Kernan, N. A., Moen, R. C., Kohn, Relationship betweensimian virus 40 D., Yu, S.-F., Karson,E., Karlsson,S., large tumor antigen expression and Zwiebel,J. A., Gilboa, E., Blaese, R. tumorformation in transgenic mice. J. M., Nienhuis,A., O’Reilly,R. J., Anderson, W. F. 1987. Expression of human Virol. 61:2029-32 44. Magli, M.C., Dick, J. E., Huszar,D., adenosine deaminase in non-human Bernstein, A., Phillips, R. A. 1987. primates after retroviral mediatedgene Modulationof gene expression in multransfer. J. Exp. Med.166:219-33

Annu. Rev. Immunol. 1988.6:581-594. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Annu. Rev. Immunol. 1988.6:581-594. Downloaded from arjournals.annualreviews.org by HINARI on 08/28/07. For personal use only.

Annual Reviews www.annualreviews.org/aronline

Annu. Rev. Immunol. 1988.6:595-628. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

Ann. Rev. Immunol.1988. 6: 595-628 Copyright©1988by AnnualReviewsInc. All rights reserved

C1 INHIBITOR AND HEREDITARY ANGIONEUROTIC EDEMA Alvin E. Davis III Divisions of Immunologyand Nephrology, Departmentof Medicine, Children’s Hospital; Departmentof Pediatrics, HarvardMedicalSchool, Boston, Massachusetts 02115 INTRODUCTION C1inhibitor (C1 INH)is a protease inhibitor involved in the regulation of several proteolytic systemsin plasma,including the complement, coagulation, fibrinolytic, and contact (kinin-forming)systems. Theimportant physiologic role of this protein is best demonstratedby the dramatic symptoms and biochemicalabnormalitiesin individuals with either hereditary C1 INHdeficiency (hereditary angioneurotic edema--HANE), the syndromeof acquired C1 INHdeficiency. Although muchhas been learned in the past several years about the structure, genetics, mechanism of action, and inhibitory spectrumof C1INH,numerousquestions in each of these areas remain unresolved. For example,the pathophysiologyof the generation of symptomsin HANE remains inadequately defined. C1 INHis clearly the only plasmaprotease inhibitor that regulates C1activation, andit is the majorinhibitor of the enzymesinvolvedin activation of the contact system.Asmighttherefore be predicted, evidenceexists for both complementand contact system activation in HANE,but the major mediator(or mediators)has not beenfully characterized. Similarly, only fragmentaryinformation is currently available regarding the specific molecular genetic defects in the C1INHgene that lead to deficiency, although the recent cloning of the geneshould rapidly yield information in this regard. Afurther exampleis providedby the fact that the C1INH moleculehas an aminoterminal extension that is heavily glycosylated 595 0732-0582/88/0410-0595502.00

Annual Reviews

Annu. Rev. Immunol. 1988.6:595-628. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

596 DAVIS comparedwith other related protease inhibitors. Thefunctional relevance of this peculiar structural feature is completelyunknown.Thecontrol of synthesis of C1 INHremains virtually an unexplored area: This is of particular importancesince plasmaC1INHlevels in patients with HANE increase in response to therapy with androgens.Thepotential regulation of C1 INHgene expression by androgens has not been investigated. Throughthe use of moderntechniques in protein chemistry, enzymology, and cell and molecular biology, these and other problemsrelated to C1 INHstructure and function andits deficiencyshouldbe solved. This review summarizesknowledgerelated to C1INH,and points out areas, including those outlined above, in whichcurrent research tools should be able to answerimportant unresolved questions. Discovery, Isolation, and Physicochemical Characterization Duringthe initial investigations that resulted in the isolation andcharacterization of C1(1-3), humanserumwasfound to contain a heat sensitive substancethat inhibited the enzymaticactivity of C1(2). This inhibitory activity, termed C1cstcrase inhibitor, was further defined by Levyand Lcpow (4) andwasfirst isolated by Pcnskyct al (5). It is clearly distinct fromother plasmaprotease inhibitors. At about the sametime, Schultzc ct al (6) purified a protein of unknown function that they namedalpha-2ncuramino-glycoprotein. It wasnot until 1969that this protein wasshown to be identical with C 1 INH(7). Detailed knowledgeof the structure of C1 INHwas delayed primarily becauseof difficulties in directly determiningthe aminoacid sequenceof so heavily glycosylated a protein. Hauptet al showedinitially in 1970 that C1 INHcontained a very large amountof carbohydrate (approximately35%by weight) (8); in fact, it is the most heavily glycosylated plasmaprotein. In agreementwith earlier work,they estimatedits molecular weightto be 104,000by analytical ultracentrifugation (5, 8); it had a low sedimentation coefficient of 3.67S. Subsequentmolecular weight determinations by sodiumdodecyl sulfate-polyacrylamide gel electrophoresiswerein agreementwith these estimates(9, 11). Its electrophoretic mobility wasthat of an alpha-2-globulin, and it had an isoelectric point of 2.7-2.8. Its extinction coefficient (E 1%,1 cm)280-1 isreported as 4.5 in this study(8) andas 3.6 in a later study(11). It consists of a single polypeptide chain (9-12), and electron microscopicanalyses revealed two domainstructure (13). These studies indicate an elongated molecule that consisted of a 33-nm-long,rod-like domainwith a diameterof 2 nm, and a terminal 4-nm-diameterglobular structure. This observedstructure is compatiblewith the low sedimentation coefficient reported (8).

Annual Reviews

Annu. Rev. Immunol. 1988.6:595-628. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

CI

INHIBITOR

597

electron micrographs of the interaction of C1inhibitor with proteaseshave beenreported. The carbohydratecompositionof C1INH(8, 11), in addition to a total content of 33%-35%, revealed an unusual pattern for a plasma protein with high contents of galactose and N-acetylgalactosamine,and a galactose-to-mannoseratio of 2: 1. The high N-acetylgalactosaminecontent (together with slightly high serine andthreonine contents) first suggested that C1INHcontainedO-linked oligosaccharideunits. It is interesting that removalof a large portion of the carbohydrate(60%of sialic acid and 20%of galactose) has no effect on C1 INHfunction (14). More recently, it has beenshownthat the underglycosylatedC 1 INHsynthesized by cultured HepG2hepatomacells in the presence of tunicamycinretains its ability to react with Cls (15). N-linkedglycosylationtherefore is not required for functional integrity of the molecule. C1INHprobably contains 20 oligosaccharideunits, for whichthe attachmentsites of 11 have beenunequivocallyidentified (16). Of these, 6 are glucosamine-based units linked to asparagineresidues within the typical Asn-X-Ser/Thr pattern; the remainderare all galactosamine-based units linked to serine or threonine residues. The suggestion that C1 INHcontained a high proportion of Olinked oligosaccharide was thus confirmed(11). Of the 20 carbohydrate attachmentsites, 17 are located within the aminoterminal 100 residues, widelyseparated fromthe reactive center (16) and outside the portion the protein that is homologous with other protease inhibitors. It is not surprising that underglycosylatedC1INHretains its inhibitory capacity. Therole of carbohydratein the function or catabolismof C1INHtherefore remainsincompletelydefined. Thefirst primarystructural informationwasreported by Harrison(11), whosequencedthe aminoterminal 40 residues of the protein. Salvesenet al (17) subsequently determined the aminoacid sequenceof the active center (fromP3 to P’9) by isolation and sequenceanalysis of the carboxyterminal peptide released from C1 INHduring complexformation with C 1 s, and by sequenceanalysis of the overlap peptide producedby limited proteolysis with Pseudomonasaeruginosa elastase. This confirmedthe previous finding that an aminoterminal threonine was detectable after complexformation between Cls and C1 INH(18), and showedthat the reactive site peptide bondis betweenan arginine and threonine residue. This work also provided additional data suggesting that C1 INHwas a member of the serpin familyof protease inhibitors (19-23). Numerous methodshave been published for the purification of C1 INH from plasma(9-12, 17, 24, 25). Themost widely used current method probablythat described in Ref. 11, whichresults in an overall yield of 70%-75%;the resulting C1 INHprotein produces a single band when

Annual Reviews

Annu. Rev. Immunol. 1988.6:595-628. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

598

DAVIS

analyzed by SDS-PAGE. Withsomepurification procedures, or following storage, a 96,000molecularweight C1INHcomponentis frequently seen, in additionto the 105,000dalton native protein (9, 11,25). This polypeptide appears to result fromproteolytic cleavage (9, 25) and/or partial deglycosylation (11). It apparentlyretains inhibitory activity against Cls and plasmin(9). C1 INHalso has been isolated from rabbit and guinea pig serum and partially characterized (26, 27). Rabbit C1INHis similar to the human proteinin size, stability, andfunctionalcharacteristics(26). Guineapig C INH,however,has a higher sedimentationcoetficient (6.1S), and functional propertiesthat differ fromthose of the human inhibitor (27, 28). INHfrom other mammalianspecies has been only incompletely characterized(29).

Cloning and SequenceAnalysis of C1 Inhibitor Several groupshaverecently reported the isolation and characterization of cDNA clones encoding C1 inhibitor, which, together with sequence analysis of the protein, haveresulted in the determinationof the complete primary structure of C1 INH(16, 30-32). These data showthat C1 INH is, as wasexpected, a memberof the serpin (33) "superfamily"of serine protease inhibitors (Figure 1). This "superfamily"contains several plasma proteaseinhibitors, includingalpha-1-antitrypsin,antithrombinIII, alpha1-antichymotrypsin,alpha-2-antiplasmin, mousecontrapsin, and heparin cofactor II. At least one cell-associated inhibitor is a memberof the group--the B-migrating endothelial-cell-type plasminogen activator inhibitor (39). In addition, there are several surprising membersof the group with no knownprotease inhibitor function: angiotensinogen, chicken ovalbumin,and chicken gene Y. The "superfamily" also contains at least twomoredistantly related proteins, onefrombarley (barley protein Z) (40) and another from cowpoxvirus, whichprobablythrough its function as an inhibitor is responsiblefor the hemorrhagic lesions inducedby this virus(41). As discussed previously, C1 INHhas a heavily glycosylated amino terminal extension such that the region of homology extends from residue 120 to the carboxy terminus of the protein. The degree of homology -betweenC1INHand the other serpins ranges from approximately20o/o 27%,whichis similar to the degree of ~aomology amongthe other members of the group (except between alpha-l-antitrypsin and alpha-l-antichymotrypsin, the genes for which are near one another on the same chromosome)(34, 35). It is thus apparent that C1 INHarose from ancestral gene common to the other membersof the serpin family. The high degree of sequence homologyamongthe serpins probably

Annual Reviews

Annu. Rev. Immunol. 1988.6:595-628. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

Cl INHIBITOR

599

indicates a similar tertiary structure. Amongthose serpins that are plasma protease inhibitors, alpha- 1-antitrypsin is the only one for whichthe crystal structure has been determined (36). This crystallographic data was obtained on alpha-l-antitrypsin that had been cleaved by chymotrypsin at its reactive site. It has not been possible to obtain suitable crystals from the native protein. A six-stranded sheet forming a large planar surface (the A sheet) is the major feature of the alpha-l-antitrypsin tertiary structure. In the native molecule, it is assumedthat the fourth strand of this sheet is bent back on itself to give a loop that joins Met-358 (at the carboxy terminal end of strand 4A) to the carboxy terminal 36 residue fragment. The reactive site, therefore, is within what must be a highly .stressed loop bridging this planar surface. The sheet strands and helixes from the structure of alpha-1-antitrypsin are labeled in Figure 1. Alignmentof the serpins based on the structure of alpha-l-antitrypsin (in which insertion of gaps in regions of defined secondary structure are penalized) gives better alignment than does the standard gap penalty alignment (Figure 1). This provides evidence that the serpins all have the samegeneral secondary and tertiary structure (33). Comparisonof C1 INHwith the other serpins in this wayindicates that it shares manyimportant structural features with the other membersof the superfamily and must therefore have a similar tertiary structure (16, 30). For example, C1 INH, at the so-called hinge region of the loop joining the reactive site, has the sequence Glu-Thr-GlyVal-Glu (residue 342-346; alpha-l-antitrypsin numbering). All the serpins have a highly conserved sequence in this region (GlxoX-Gly-X-Glu).Also, as would be expected, the strands forming the planar surface (the A sheet) that are immediately adjacent to the loop containing the reactive site (strands A3 and A5) are highly conserved amongall the serpins, including C1 INH. Another example is the fact that C1 INH has conserved the residues analagous to those in alpha-1-antitrypsin that are involved in salt bridges between areas of secondary structure (glu-264, 1ys-387 and glu342, lys-290). In C1 INH, arginine residues have replaced the two lysines; this has occurred in several other serpins. Consistent also with these interpretations is the fact that the regions of least conservation amongthe serpins are those that mustbe on the surface of the moleculeif their tertiary structures are similar to alpha-1-antitrypsin (the aminoterminal ends of helixes A and F; helixes C and D; sheet A, strands 1, 2 and the carboxy terminal end of strand A; and sheet C, strands 1 and 2 (16). Analysis of a full-length eDNAclone for C1 INHindicates that it is synthesized with an aminoterminal 22-residue signal peptide (16). It is typical signal peptide with a basic residue near its amino terminus, a hydrophobic core region, and a small amino acid prior to the signal cleavage site. As was alluded to earlier, most of the oligosaccharide units

Annual Reviews

600

DAVIS NPNATSSSSQDPESLQDRGEGKVATTVISKMLFVEPILEVSSLPT~STT AAT AAC GSKGPLDQLEKGGETAQSADpQWEQLNNK~L£MPLLP AAP AGT CNT OVA BPAI

Annu. Rev. Immunol. 1988.6:595-628. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

AAT AAC

AGT CNT CGY OVA

NSATKITANTTDEP%~QPTTEPTTQPTIQPTQPTTQLpTDSPTQPTTGSF EDPQGD NSPLD HGSPVDICTAKPRDIPMNPMCIYRSPEKKAT ADFHKENTVTNDWIPEGEEDDDYLDLEK~FSEDDDY~DIVDSLSVSPTD$ NQEQVSPLTLLKLGNQEPGGQ yIHPFHLVIHNESTCEQLAKANAGKPKDPTF~APIQAKTSPVDEKALQD

i0

20

30

40

5O

AAT AAC

AAQKTDTSHHDODHPTFNKITPNLAEFAFSLYRQLAHQSNS--TNIFFSP EENLTQPENQDRGTHVDLGLASA~FAFSLYKQLVLKALD--K/~41FSP

HC II AAP AGT CNT

DVSAGNILQLFHGKSRIQRLNILNAKFAFNLYRVLKDQ~T-FDNIFIAP TALKSPPGVCSRDPTPEQTHHLARAMMAFTADLFSLVAQTSTCSNLILSP QLVLVAAKLDTEDKLRAA~GMLANFLGFRIYGMHSELWGV~HGATVLSP

OVA BPAI

GSIGAASMEFCFDVFKELKVHHAN--ENIFYCP VHHPPSYVAHLASDFGVRVFQQVAQASKD--F/~V~FS~

C1 INH AAT AAC AT IiI HC II AAP AGT CNT CGY OVA BPAI

90 60 70 80 CVHQALK FSIASLLSQVLLGAGQNTKTNLESILSYPKD?T .......... PEAQIHE V$IATAF~AMLSLGTKADTHDEILEGLNFN-LTQI ......... LSISTALAFLSLGAHNTTLTEILKASSSP-HGDL ......... LRQKFT~ TSDQIHF LSISTAFAMTKLGACNDTLQQLMEVFKFDTISEK ......... VGISTAMGMISLGLKGETHEQVHSILHFKDFVNASSKY .....EITTIHN GP LSVALALSHLALGAQNHTLQ~LQQVLHAGS .................. TAVFGTLASLYLGALDHTADRLQAILGVPWKDKNCTSRLDAH-KVLSALQ LSILTALAMVYLGARGNTESQMKKVLHFDSITGAGSTTDSQCGSSEYVHN IAIMSALA~LGAKDSTRTQINKV~RFDKLPGFGDS~EAQCGTSV~HS ---~ .........GMAP YGVASVLAMLQLTTGGE~QQIQAAMGFKIDDK ix D ) i00

AAC AT iII HC II AAP AGT CNT CGY OVA BPAI

( ShA,St2) ii0 120

( Helix E I~0

140

SFQHLRAPSISSSDE--LQLSMGNAMFVKEQLSLLDRFTEDAKRLYGSEA FFAKLNCRLYRKANK-SSKLVSANRLFGDKSLTFNETYQDISEL~AKL LFRKLTHRLFRRNFG--¥TLRSVNDLYIQKQFPILLDFK~
C1 ~NH AAT AAC

A,Stl)( Meli~F ) [ ShA, 150 160 170 180 R .... VLSNNSDANLELIN~%4V~J(NTNNKISRLL--DSLPSDTRLVLLNA FTV--NFGDTEEAKKQ-INDYVEKGTQGKIVDLV--KELDRDTVFALV~ FAT--DFQDSAAAKKL-INDY%l~NGTRGKITDLI--KDPDSQ%~VLVNY

HC II AAP AGT CNT CGY OVA BPAI

QIA--DFSDPAFISKT--NNHIMKLTKGLIKDAL--ENIDPATQ~ILNC V .... SLTGKQEDDLANINQWVKEATEGKIQEFL--$GLPEDTVLLLLNA LPRSLDFTELDVAAEK-IDRFMQAVTGWKTGCSL--MGASVDSTLAFNTY VVLVNy EEV--NFKTAAEEARQLINSW~EKETNGQIKDLLV$S$IDFGTTMVFINT EPI--NFOTAAD~ARELINEWVESQTNGIIRNVL~PSSVDSQTAMVLVNA KQV--DFSEVERARFI-INDWVKTHTKGMISNLLGKGAVDQLTRLVLVNA

Annual Reviews C1 INHIBITOR

AAC

Annu. Rev. Immunol. 1988.6:595-628. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

AAC

AGT CNT OVA

AAT AAC

AGT OVA

190 200 210 220 230 ~ YLSAKWKTT?DPKKTRMEpFHFKNS -V~ KVP~SKKYPV- -AHFID~T ~ FFKGKWERP?E~TEEEDFHVDQV~VPMMKRLGMF - - -N I QHCKK ~ FFKAKWEMPFDPQDTHQSRFYLSKKKWVMVP~4SLHHLTI - - PYFREEE I YFKGLWKSKFSPENTRKELF~GESCSAS~IYQEGKF - - - RYRRVAE I YFKGSWVNKFPVEMTH/~HNFRLNEREVVKVS~QTKGNF - - - LAANDQE ~ HFQGF~KFDPSLTQRD~FHLDEQ~TVPVE~ARTY~L - -RWFLLEQ VHFQGKMK-G?$LLAEPQE- FWVDNSTSVSVPMLSGMGTF- -QHWSDIQ I NFKGKWKIS ?DPQDTFESEFYLDEKRSVKVP~AMKLLTT-RHFRDEE I YFKGIWK I AFNTEDTREMPFSMTKEESKPVQ~4C~4NSF- -NVATLPA I VFKGLWEKAFKDEDTQAMPFRVTEQESKpVQ~4YQI GLF- - -RVASMAS LYFNGQWKTPFPDSSTHRRLFHKSDGSTVSVP~4AQTNKFN~TEFTTPDG !ShB.St2) (ShB,St3) (HelixG)(Helix 240 250 260 270 LKAKVGQLQLSH-NLSLVILVPQNLKHRLEDMEQALSPSVFKAIMEKL-E LSSWVLLMKYLG-NATAIFFLPD--EGKLQHLENELTHDIITKFLENEoLSCTVVELKYTG-NASALFILPD--QD~MEEVEAMLLPETLKRWP.DSL-E -GTQVLELPFKGDDIT~F4LILPKP-EKSLAKVEKELTPEVLQEWLDEL-LDCDILQLEYVG-GISMLIWPHK-MSGMKTLEAQLTPRWERWQKSM-PEIQVAHFPFKN-NMSF~VLVPTHFEWNVSQVLA ...... NLSWDTLHP~ DNFSVTQVPFTE-SACLLLIQPH~-ASDLDKVEGLTFQQNSL~-LSCSVLELKYTG-NASALL~L~D--QGRMQQVEASLQPETLRKWRKTL-EK/~KILELPYASGDLSMLVLLPDE-VEGLER~EKT~NFDKLRE~S’I~/4-A E~4KILELPFASGTMSMLVLLPDE-VSGLEQLESIINFEKLTEWTSSN-V HYYDILELPYHGDTLSMFIAAPYEKEVpLSALTNILSAQLISHWKGNM-280 2~0 300 310 320 MSKFQPTLLTbPRIKVTTSQDM-LSIMEKLEFFD-FSYDLN-LCGLTEDP --DRRSASLHLPKLSITGTYDL-KSVLGQLGITKVFSNGAD-LSGVTEEA F--REIGELYLPKFSI~RDYNL-NDILLQLGIEEAFTS~-LSGITGAR --EEMMLWHMPRFRIEDGFSL-KEQLQDMGLVDLFSPEKSKLPGIVAEG --TNRTREVLLPKFKLEK~L-VESLKLMGIRMLFDKNGNMA-GIEDQR LVWERPTKVRLFKLYLKHQMDL-VATLS~LGLQELFQAPD--LRG15EQS --SPRTIHLTMPQLVLQGSYDL-QDLLAQAELPAILHTELN-LQKLSNDR F-PSQIEELNLPKFSIASNYRLEEDVLPEMGIKEVFTEQAD-LSGIIETK M-AKKSMKVYLPRMKIEEKYNL-TSILMALGMTDLFSRSA~-LTG~$SVD M-EERKIKVYLP~MK/4EEKYNL-TSVLMAMGITDVFS~SAN-LSGISSAE --TRLPRLLVLPKFSLETEVDL-RKPLENLGMTDMFRQFQADFTSLSDQE

OVA BPAI

( 5hA, SiS }( ShA, S~4 } ( ShC,Stl 340 350 *360 --DLQVSAMQHQTVLELTETGVEAAAASAISVA--RTLLV .....FEVQQ --PLKLS~VHKAVLTIDEKGTEAAGAMFLEAIP-MSIP?E ....VKFNK --NLAVSQWHKVVSDVFEEGTEASAATAVKITL-LSALVETRTI%FRFNR RDDLYVSDAFHKAFLEVNEEGSEAAASTAV~IAG-RSLNPN’RVTFKANR ---IAIDLFKMQGTITVNEEGTQATTVTTVGFMP-LSTQVR ....FTVDR ---LWSGVQMQSTLELSEVGVEAAAAT$1AMS--RMSLSS ....FAVOR ---IRVGEVLMSIFFEL-EADEREPTES~QLNK-PEVLE .....VTLNR --KLSVSQWMKAVLDVAETGTEAAAATGVIGGIRKAILPA ....VMFMR --NLMISDAVHGVFMEVNEEGTEATGSTGAIGNIKHSLELEE---FR~H -oSLKISQAVHAAHAEINEAGREWGSAEAGVDA-ASVS-EE---FRADH --PLHVAQALQKVKIEVNESGTVASSSTAVIVSA’RMApEE .... IIMDR (ShB,S~4)( ShB,$~5 370 380 390 PFLFMLWDQQHKFpVFMGRVYDPRA pFVFLMIEQNTKSPLFMGK~PTQK PFLMIIVPTDTQNIFFMSKVTNPKQA PFLVFIREVpLNTIIFMGRVA~PCVK PFLFLIYEHRT$CLLFMGRVANPSR$ PFLFFIFEDTTGLPLFVGSVRNPNPSAPRELKEQQDSPGNKDFLQSLKGF PFLFAV~DQSATALMFLGRVANPLSTA PFLFVIY~TSAQSILFMAKV14NPK PFLFFIRYNPTNAILFFGR~WSP PFLFCIKHIATNAVLFFGRCVSP PFLFV~RHNPTGTVLFMGQVMEp

AAP

PRGDKLFGPDLKLVPPMEEDYPQFG$ PK

AAP AGT cNT CGY OVA

AAT AAC AAP AGT CNT

601

Figure I The amino acid sequence of C1 inhibitor comparedwith the sequences of the other serpins. Theabbreviationsandreferences for the sequencesare as follows: CI inhibitor (C1 INH)(16); alpha-l-antitrypsin (AAT)(19, 33, 37); alpha-l-antichymotrypsin (21, 37); antithrombinIII (ATIII) (37); heparin cofactor II (HCII) (22); alpha-2-antiplasmin (AAP)(23); angiotensinogen (AGT) (37); mouse contrapsin (CNT)(21); chicken (CGY)(38); chicken ovalbumin(OVA)(37); beta-endothelial cell-plasminogen activator inhibitor (BPAI)(39). A dash signifies a gap in the sequence. Helices andsheet strands the crystal structure of cleaved alpha-l-antitrypsin are indicated above the sequences(16, 36). Residuenumberingis that of alpha-l-antitrypsin. The P1-PI’ peptide bondis indicated by an asterisk.

Annual Reviews

Annu. Rev. Immunol. 1988.6:595-628. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

602 DAVIS (17 of 20) in C1 INHare located in the aminoterminal region of the molecule.Of these 10 are within a 35-residue region in whichsequences similar to the sequenceGly-Pro-Thr-Throccur seven times; this repeat results fromtandemrepetitions of a 12-basenucleotide sequence(16). addition, the disulfide bridging pattern in C1INHhas been determined (four cysteineresiduesin a 1-4, 2-3 pattern). Thepattern is different from those of alpha-1-antitrypsin and alpha-1-antichymotrypsin (each of which have one cysteine) but showssomesimilarity to that of antithrombinIII (six cysteinesin a 1-4, 2-3, 5-6 pattern) (16). Southernblot analysis of DNA from rodent-humansomatic cell hybrids has localized the C1 INHgene to subregion 11.2-q13 of humanchromosome11 (16, 30). Noother serpin is knownto be encodedon this chromosome:the alpha-l-antitrypsin geneis on chromosome 14 (q32.1) (34, 42), as is that for alpha-l-antichymotrypsin(35), while the locus for antithrombin III is on humanchromosome 1 (q23~125)(43, 44). One polymorphismhas been identified in the normal C1 INHgene, whichwas initially found by comparisonof protein and cDNA sequencing data (16). At residue 458, a valine was identified by protein sequence analysis, while the cDNA sequencecodedfor a methionineat this position. The valine variant codes for an HgiAIrecognition sequence, and an HgiAI RFLPwas identified. The polymorphismhas two alleles, which produce 0.7 and 0.4 kb bands, with Mendelianinheritance and frequencies of 0.32 and 0.68, respectively. No other RFLPshave been identified in the C1 INHgenein normalindividuals (16, 45). Genomicclones for C1 INHhave recently been isolated and characterized (S. C. Book,personal communication). Beginningwith an intron that is 22 bases upstreamfrom the initiator ATGcodon, there are a total of seven exons and seven introns. The entire geneis approximately17 kilobases in length. Althoughthe geneis larger than those encodingthe other serpins, at least one intron is located at a position homologous to introns found in antithrombinIII, alpha-l-antitrypsin and angiotensinogen. In addition, the introns mapto locations betweendefined domainsin alpha1-antitrypsin. C1 Synthesis Theprimarysite of synthesis of C1INHin vivo is probablythe liver (46, 47). This was shownby immunofluorescencestudies of hepatic parenchymalcells with antibody to C1INH(6). Subsequently, the hepatoma cell line, HepG2, was shownto synthesize C1INH(15, 48). Twoforms of C1 INHproteins were secreted, one of whichappearedvery slightly | larger than C1INHisolated from plasma; the other was a minor componentwith a molecularweightof 90,000(15). Thelarger form wasalso

Annual Reviews

Annu. Rev. Immunol. 1988.6:595-628. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

CI

INHIBITOR

603

found in small quantities intracellularly together with a major Mr 80,000 form. This Mr 80,000form accumulatedwhencells were incubatedat 23°C or whenthe synthesis studies were done in the presence of monensin (whichinhibits protein secretionand late glycosylation).In the presence tunicamycin(which blocks N-linked glycosylation), an apparent decrease in size of both the secreted and intracellular forms was observed. Both thus contain N-linkedoligosaccharide, and this indicates that complete glycosylation is not required for secretion. Thelarge secreted component probably represents the fully glycosylated C1 INHprotein, while the intracellular formis partially glycosylated.Theseconclusionswereinferred from the sensitivity to endoglycosidases:extracellular C1INHwassensitive to endoglycosidase F but resistant to endoglycosidase H(indicating the presenceof complextype N-linkedcarbohydrate), while the intracellular protein wassensitive to both. In addition, cell-free translation studies (using normal humanliver mRNA) resulted in a C1 INHpolypeptide apparent Mr 60,000 (31), whichis similar to the size predicted from the aminoacid sequence(16). C1 INH, like manyother complementcomponentsand like alpha-1antitrypsin (49) is also synthesized by cultured humanperipheral blood monocytes(45, 50, 51), and by the humanmonocyte-likecell line, U937 (52). In one of these reports, C1INHsynthesis was detected after minimum of one weekin culture (50) (after the cells becomemacrophagelike), while in another, synthesis wasdetected only after stimulationwith lymphokines(51). Cicardi et al, however, found that C1INHwas synthesized fromthe first day in culture (45). Part of these differences probablydue to variation in the culture techniques. For example,in our studies, the mononuclear cells are incubatedovernightin autologousserum prior to plating the cells (45), whilein the other studies this wasnot done. This mayvery well enhancesynthesis. Wehave also detected specific C1 INHmRNA in freshly isolated monocytesanalyzed within 1-2 hr after blood wasdrawn(M. Cicardi, A. E. Davis, unpublisheddata). It is thus not surprisingthat specific synthesismightbe detectedveryearly in culture. This further suggests that synthesis of C1INHis a physiologic property of circulating monocytes,as also maybe true of alpha-l-antitrypsin (49). Thesynthesis of these protease inhibitors by circulating monocytesmay be important in the local regulation of inflammatoryphenomena. Immunoprecipitationof metabolically labeled C1 INHfrom monocyte culture supernatants showedresults basically similar to those described above for C1 INHsynthesized by cultured HepG2 ceils (15). A C1 INH moleculeindistinguishable in size fromthat foundin plasmais secreted into culture supernatants, while small amountsof this component, together with a major underglycosylatedform with a molecularweightof approxi-

Annual Reviews 604

DAVIS

Annu. Rev. Immunol. 1988.6:595-628. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

mately 80,000 are found intracellularly (45, 50, 51). The synthesis C1 INHby cultured monocytes and by hepatocytes thus appears to be qualitatively similar. In addition to the above, placenta is apparently capable of C1 INH synthesis (47). Megakaryocytesmayalso synthesize C 1 INH. Platelet alpha granules contain C1 INH, which is secreted along with other alpha granule contents during platelet activation (53, 54). Mechanism of C1 INH Function C1 INHis able to inactivate several plasma serine proteases, including the Clr and Cls subcomponentsof C1 (2, 9, 55), kallikrein (55, 56), plasmin (9, 55), coagulation factors XIa and XIIa (57), and the enzymatically active fragments derived from factor XIIa (factor XIIf) (58). It is the only known inhibitor of Clr and Cls (59, 60), while each of the other proteases also may be inactivated by other plasma protease inhibitors. Sim et al (59) incubated enzymatically active ~25I-labeled Clr and Cls in plasma and demonstrated the formation of radiolabeled high molecular weight complexes by gel filtration and by SDS-polyacrylamidegel electrophoresis. All of the complexed radioactivity was removed by immunoabsorption with anti-C1 INH antibody, thus demonstrating that C1 INH was the major plasma inhibitor of Clr and Cls. Ziccardi further (60) showed that the kinetics of inactivation of Clr and Cls were the same in serum as in purified mixtures of C1 INHwith the active enzymes. C1 INH does not inhibit but is cleaved at varying sites, by several other proteases including trypsin (8, 9), humanleukocyte elastase (61), several snake venom teases (62), and Pseudomonasaeruginosa protease and elastase (63). of these proteases inactivate C1 INH, in somecases due to cleavage at or near the active site of the inhibitor (17). The snake venomproteases, however, do not universally result in inactivation. The Elapid venoms produce an active C1 INHspecies that is reduced in size from Mr 105,000 to Mr 89,000. Crotolid, Viperid, and Colubrid venomsproduce an intermediate of the same size that retains activity; this product is further degraded to an Mr 86,000 inactive species (62). With the Crotalus atrox protease, the active Mr 89,000 product results from removalof a 36 residue aminoterminal peptide (17), thus showingthat at least this aminoterminal segment is not required for function. C1 inhibitor almost certainly functions by the same general mechanism as several of the other membersof the serpin family, of which alpha-1antitrypsin is the prototypical member.Inhibition of the susceptible protease occurs via binding to a region (the reactive site) within the inhibitor that mimics the substrate (64, 65). At the reactive site is an amino acid residue specifically recognizedby the substrate binding site of the protease

Annual Reviews CI INHIBITOR

605

Annu. Rev. Immunol. 1988.6:595-628. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

that is susceptible to inhibition; this residue is termedthe P~residue (66). The residue carboxy terminal to the P~ residue is termed P~. The peptide bond between these two amino acid residues is cleaved during formation of the complexbetweenthe protease and the inhibitor. Each of the inhibitors in the serpin family has a single active site and thus forms equimolar complexeswith its target proteases. The complexesformed are extremely stable and are not dissociated by denaturants, which indicates that the interaction must be covalent and must result from formation of a stable acyl-enzymederivative.

Interaction with Active Clr and Cls C1 INHreacts with enzymatically active Clr and Cls to form equimolar complexes that are not dissociated by heat, SDS(9, 67-69), guanidine, urea, or low pH (25). However, the complexes are slowly dissociated alkaline pH (25, 59) and are completely dissociated by treatment with hydroxylamine (59). The covalent complex therefore appears to involve an ester linkage. Formation of the complexinactivates the activity of the enzymes(9, 67, 69). The interaction is via the light chains of Clr and Cls, which are the active-site containing chains. Analysis of the complexesby SDS-polyacrylamidegel electrophoresis under reducing conditions reveals a band with a molecular weight consistent in size with that of a single molecule of C1 INHplus a single molecule of the light chain of Clr and Cls. In addition, a band that is the samesize as the heavychain of activated Clr or Cls is also present (9, 59, 67). Previous inactivation of Clr or Cls with diisopropylfluorphosphate prevents complex formation with C1 INH, further indicating that the interaction is via the active site of the protease (69-72). A small carboxy-terminal peptide is released from C1 inhibitor during complex formation with Cls (73). As described earlier, sequence analysis of this peptide and an overlap peptide identified the P~-P~residues as an arginine and a threonine, respectively (17, 18). The mechanism inhibition by C1 INH, thus, is entirely consistent with that of other members of the family. The reaction of C1 INH with activated Clr and Cls follows second order kinetics. C1 INHreacts more slowly with Clr than with Cls, with rate constants of 2.8 x 103 M-~sec- t and 1.2 x 104 M-t sec- ~, respectively (74). A recent report using several different methods to detect complex formation and loss of enzymatic activity found a rate constant of 6.0 x 104 M-~ sec -~ for the interaction of Cls with C1 INH (75). The rate interaction of Cls with C1 INHis not affected by the presence of EDTA or calcium (74, 75), while the Clr-C1 INHinteraction is only half as fast in EDTAas in calcium (74). The Clr reaction is also more sensitive temperature and ionic strength. In addition, the activation energy for the

Annual Reviews

Annu. Rev. Immunol. 1988.6:595-628. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

606

DAVIS

binding of activated Clr by C1 INHis greater (44.3 kcal/mol) than for the binding of C1 s (11.0-11.7 kcal/mol) (74, 75). The equilibriur~ constants -7 for reaction of C1 INHwith Clr and Cls are nearly the same (1.21 x 10 and 9.6 x 10-8 M, respectively) (74). The rate of reaction between C1 INHand activated Cls and, to a lesser extent, Clr, is enhanced by heparin (25, 74, 76-78). Sim et al (74), example, found that Cls-C1 INH complex formation was enhanced 1415-fold in the presence of heparin. Lennick et al (75) found a similar enhancement in the rate of complex formation and showed that heparin binds with similar affinity to C 1 INH,C 1 s and to the bimolecularcomplex. The anticomplementaryactivity of heparin .(79, 80) is probably primarily due to this effect on Clr and Cls inhibition since muchlarger quantities of heparin are required to inhibit complementactivation if C1 INHis functionally removed from serum by the addition of antibody to C1 INH (77). It has also been reported that formation of a C1 INHderivative that presumablyis cleaved at its active site but is not complexedwith Cls is enhancedin the presence of heparin (73). It is unclea~ if this derivative represents C1 INHthat is modified before being trapped in a stable complex,if it is released from the complex,or if it results from an artifact related to SDS-polyacrylamidegel electrophoresis. A modified C1 INHof similar size has been found in the plasma of patients with hereditary and acquired angioneurotic edema(see discussion below). Interaction with CoatTulation and Contact System Proteases As with activated Clr and Cls, C1 INHforms stable, apparently covalent, bimolecular complexeswith each of the other proteases that it inhibits (9, 81-86). Small amounts of modified C1 INHwith an Mr of 95,000 are also observed, as seen during the interaction with Clr and Cls. In addition, the complexwith plasmin is further degraded by excess plasmin to give an additional inactive product (9). Heparin does not appear to have a significant effect on the inhibition of kallikrein by C1 INH(65). The rate constants for the interaction of C1 INHwith these other proteases are in the same range as those for complex formation with Clr and Cls. The value for plasma kallikrein varies from 1.7-4.5 × 104 M- 1 sec- ~ depending on temperature (81, 82); the value for the reaction with factor XIa 6.7 × 102 M-~ sec -~ (87); those for the reaction with factors XIIa and XIIf are intermediate between the two (3.7 and 3.1 × 10 3 M-1 -~, sec respectively) (83, 84). Thesekinetic studies also provideddata that strongly indicate that only the portion of the protease molecules containing the active sites are involved in complex formation with CI INH(86). The similar rate constants for inactivation of factors XIIa and XIIf are con-

Annual Reviews

Annu. Rev. Immunol. 1988.6:595-628. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

Cl INHIBITOR

607

sistent with this suggestion.In addition, intact twochainkallikrein andits isolated light chainare inactivatedat the samerate (82), as is also a three chain form of kallikrein (85). It has been suggestedthat high molecular weightkininogen,whichcirculates partially boundto kallikrein, exerts a protective effect on C1INHinactivation of kallikrein (81, 88). Twoother subsequent reports, however, were unable to showany effect by high molecularweightkininogenon kallikrein inactivation (82, 89). Complexformation between C1 INHand its target proteases results both in the maskingof epitopes on the enzyme(69, 90) and in the generation of neoantigenicdeterminantson the inhibitor-protease complex.Activated Clr, for example,fails to react with mostantisera to Clr whenit is complexedwith C1INH(90). Thus, activation of C1in serumresults an apparentdecreasein the quantity of Clr antigen. Asimilar phenomenon was shownusing someantisera to Cls (90). Thus, most antisera to Clr are probablydirected towardthe light chain or the portion of the light chain involved in binding to the inhibitors, while the antigenic determinants on Cls must be spread through more of the molecule. The disappearanceof Clr as a result of complexformation has been used as the basis of assays to quantitate C1activation in various disease states and as a meansof quantitating C1 INHfunction in serumof patients with angioneurotic edema(91-94). For this latter assay, C1is activated serumby the addition of aggregatedIgG, andresidual C l r antigen is then quantitated by radial immunodiffusion (94). In normalserum,the apparent Clr concentration decreases to nearly undetectable levels, while in C1 INH-deficientserum, little apparentdecreaseis observed. Complexformation between C1INHand plasma kallikrein results in the appearance of a neoantigen on the complex(95), as was suggested by earlier studies of the reaction between thrombin or factor Xa and antithrombinIII, and betweenplasmin and alpha-2-antiplasmin(96-99). A monoclonalantibody was described that reacted with the complexand with the Mr95,000proteolytically modifiedC1INH,but that did not react with either of the native proteins. This and similar antibodies to other complexes mightbe of use in the evaluationof diseases in whichactivation of enzymesinactivated by C 1 INH(or other protease inhibitors) is thought to occur. The Role of C1 INH in Control of Different Proteolytic Systems As pointedout earlier, C 1 INHis the only knownplasmaprotease inhibitor of activated Clr and Cls (59, 60) and is thus of paramountimportance control of classical complement pathwayactivation (see further discussion below). However,its physiologic role in the control of other protease

Annual Reviews

Annu. Rev. Immunol. 1988.6:595-628. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

608 DAVIS systemshas beenmoredifficult to assess. This difficulty results largely from the fact that all of the other susceptible proteases mayalso be inactivated by one or moreother inhibitors. As pointed out by Travis & Salvesen(65), the availablekinetic data are not of assistancein this regard, since the reported rates of inhibition are all extremelyslowin comparison with other protease-proteaseinhibitor interactions. Anotherapproachto these questions is the evaluation of inhibitor function in wholeserumor plasma. C1INHis clearly of major importancein control of the plasma kinin-formingsystem. The first suggestion that C1INHmight be important in the inactivationof kallikrein wasprovidedby the early findingthat the plasmaof patients with hereditary angioneuroticedemaweredeficient in the ability to inhibit kallikrein as comparedwith normalplasma(100). Subsequentstudies comparingthe inhibition of kallikrein by normaland C1 INH-deficient plasma concluded that C1 INHwas the major plasma inhibitor of kallikrein (56, 101). Anotherreport indicated that although alpha-2-macroglobulinand C1INHin plasma both inhibited kallikrein, the bulk of the inhibition was due to C1INH(102). Morerecent quantitative kinetic analysis of the inactivation of purified kallikrein by normal plasma found that C1 INHprovided 42%of the inhibitory capacity in plasma, while alpha-2-macroglobulinprovided 50%,and all other inhibitors accountedfor 8%of the inactivation (103). Additionof radiolabeled kallikrein to normalplasmaand analysis by gel filtration showedthat 52% of the enzymewas complexed with C1 INHand 35%48% with alpha-2macroglobulin (103, 104). Bycontrast, in C 1 INHdeficient plasma,greater than 90%of the kallikrein was complexedwith alpha-2-macroglobulin (103). Therate of kallikrein inactivation wasmarkedlyslower than normal in C 1 INHdeficient plasma,but only slightly decreasedin alpha-2-macroglobulin deficient plasma(104). Using an enzyme-linkedimmunosorbent assay, C1 INH-kallikrein complexformation has been quantitated in plasma,both after the addition of exogenouskallikrein and after contact system activation by the addition of Hagemanfactor fragment (factor XIIf) or kaolin (105, 106). C1 INHbound 57%of exogenously added kallikrein and 84%of the kallikrein resulting from contact systemactivation. Kinetic data suggest that C1 INH is even more important for the inactivation of factors XIIa and XIIf, accounting for approximately90% of the inactivation of both (83, 84). It was also shownby SDS-polyacrylamidegel electrophoresis that the majorcomplexesformedwerethose with C1 INH.Factor XIa, on the other hand, as wouldbe expected from the kinetic data usingpurified proteins, is primarilyinactivated by alpha1-antitrypsin, and C1 INHonly accounts for a very small proportion of its inhibition (87). Plasmin,whichin purified systemsreacts with C1INH

Annual Reviews CI

INHIBITOR

609

at rates similar to those of Cls andkallikrein (9, 81), doesnot seemto significantly inactivatedby C 1 INHin plasma(107, 108). Its majorinhibitors in plasmaare alpha-2-antiplasminand alpha-2-macroglobulin(107,

Annu. Rev. Immunol. 1988.6:595-628. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

108). Control of C1 Activity Macromolecular C1 is a reversible calcium-dependentcomplexconsisting of one molecule of Clq and two molecules each of proenzymesClr and Cls (Clr2s2) (109-115). Binding of C1, via its Clq subcomponent,leads to C1activation, whichis characterized by conversionof the Clr and Cls subcomponents to their active forms. Theseoccur by limited proteolysis of single chain zymogen Clr andCls to twochain proteases (67, 116, 117). Activationmayalso occurinefficiently in the absenceof an activator (I 18). C1INHprevents this autoactivation of fluid phaseC1but does not prevent C1activation in the presenceof a C1activator (119). It does, however, inactivate C1onceit has beenactivated, by binding to the C lr and C ls subcomponentsin the samemanneras with the isolated enzyme(69, 72, 90, 120). The reaction rate of CI INHwith Cls within activated C1 is similar to the rate with the free enzyme.Clr, however,reacts morereadily with C1 INHwhenit is within the activated complex. The Clr within activated C1, however,still reacts moreslowlythan doesCls (69, 72, 74). In addition to binding to the Clr and Cls within activated C1, C1INH also promotesthe dissociation of C1, resulting in the release of C1INHClroCls-C1INHcomplexesfrom the Clq. Laurell et al (121) first recognized these complexesin both normalserumand in serumof patients with hereditary angioneurotic edema. These and other authors then showed that these complexesappearedafter C1activation in seruminduced by any of a numberof classical pathwayactivators (91, 122). Ziccardi Cooper (123) demonstrated that each Clr and Cls molecule was bound to a C1INHmolecule. Froma sedimentation rate of 9S and a diffusion coefficient of 2.3 × 10-7 cm2 see-1, they calculated a molecularweightof 330,000for the complex,whichis consistent with a complexmadeup of two molecules of C1 INHand one molecule each of activated Clr and Cls. Sim et al (72) examined C1 INH-inducedC1 dissociation from ovalbumin-antiovalbuminimmunecomplexesin serum; they showedthat release varied linearly with C1INHconcentration, wasdependenton ionic strength, and that little if any Clq was released from the complexes. Treatment of activated C1 with diisopropylfluorophosphate abrogated release and complexformation with C1 INH, while treatment of either activated Clr or Cls resulted in release on addition of C1 INH. As expected, complex formation occurred only with the componentnot treated with diisopropylfluorophosphate. AlthoughCls complexedmore

Annual Reviews

Annu. Rev. Immunol. 1988.6:595-628. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

610 DAVIS rapidly with C1INH,it is the interaction with Clr that is mostimportant in causing release (72). The Clq that remains bound to the immune complexmaybe involved in binding to Clq receptors that have been identified on B lymphocytes,monocytes,null cells, polymorphonuclear leukocytes,platelets, and endothelialcells (124-135). As a result of these actions of C1INH,the function of activated CI is very efficiently controlled. Activationof C 1 by limiting amountsof immune complexes,aggregated IgG, or nonimmune activators resulted in rapid complex formation between C1 INH, and Clr and Cls (60). Results were identical in either serumor in mixtures of the isolated proteins at physiologic concentration. Consequently, C4 and C2 turnover were diminished. Doekeset al (136) also showedthat C1 INHdiminished the C4-consuming ability of C1activated with soluble IgG aggregates. As comparedwith the absenceof C1INH,at physiologic C1INH:C1ratios, an eight-to-ten-fold increase in aggregate concentrationwasrequired to maintain constant C4 consumption. In addition, C1 INHreduced the maximum C4consumptionthat could be induced, and this wasparticularly true with smalleraggregates.Thus,with reducedlevels of C 1 INH,classical pathway activation by immunecomplexes (as assessed by C4 and C2 consumption)occurs moreefficiently and is inducedwith small complexes that do not induce activation in the presence of normal C1 INHconcentrations. The measurementof C1 INH-Clr-Cls-C1INH complexesin serum has beenused as a meansof analyzingclassical pathwayactivation in various disease states. Originally, immuno-diffusion(91), crossed immunoelectrophoresis (122), and electroimmunoassay (137) were used, but recently sensitive radioimmunoassay(138) and enzyme-linkedimmunosorbent assays havealso been reported (139). Theseassays have beenused to detect classical pathwayactivation in hereditary augioneuroticedema (121), in systemiclupus erythematosus,and in glomerulonephritis(140). As mentionedabove, C1 INHprevents the spontaneous autoactivation of C1, whichexplains whythe autoactivation that occurswith reconstituted mixtures of purified Clq and zymogenClr and Cls does not occur in normalserum(118, 119). The addition of C1INHat levels ranging from 0.35 to 2.0 times the physiologic level to purified macromolecularC1 blockedautoactivation and activation by nonimmune C 1 activators (119). At 37°C, immunecomplex-induced activation wasunaffected, but at 20°C, C1activation wasinhibited. Othershave foundsimilar results (141), but Folkerd et al reported that C1 INHalso prevented C1 activation by immune complexes(142). Weak,specific binding of C 1 INHto unactivated C1 (Clr and Cls in zymogenforms) was demonstrated on the surface sensitized sheep erythrocytes. No covalent complexesbetween C1 INH

Annual Reviews

Annu. Rev. Immunol. 1988.6:595-628. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

C1 INHIBITOR

611

and Clr or Cls were observed.This function, therefore, is very different fromthe protease inhibitory function of C1INH.It has not been determinedwhetherthis activity is mediatedvia an interaction at the active center of C 1 INH,or if it is mediatedby anotherdomainwithinthe protein.. Thesefindings have been strengthenedby the subsequentdemonstrationof direct fluid phase interaction betweennative C1and C1 INH(143). ultracentrifugation in sucrosedensity gradients, in the presenceof excess C1, C1INHcosedimentedwith native C1 with an S-rate of 16S. Nitrophenylguanidinobenzoate (a reversible inhibitor of C1activation) competed with C1INHfor binding to C1, suggestingthat C1INHis reversibly binding to the active site of zymogenClr and/or Cls. There are no data to indicate whetherC1INHis binding to native C1or to an intermediate form of uncleavedC1 with altered conformation, as has been suggested (119, 144-146). Anintermediate form of Clr has been directly demonstrated (145, 146). As Ziccardi has suggested (147), C1 autoactivation secondary to diminished C1 INHlevels mayaccount for the classical pathwayactivation that is characteristic of C 1 INHdeficiency(hereditary and acquiredangioneuroticedema,see below). Additionalfactors involved maybe the moreefficient classical pathwayactivation by small immune complexesor low concentrations of immunecomplexesthat occur with low CI INHlevels (136), or via interaction with proteases of the contact activation system. Hereditary

Angioneurotic

Edema

In his classic description in 1888(148), Osier pointed out that HANE had been described and namedseveral years earlier by Quincke(149), and several other reports hadappearedin the literature. Theseearly descriptions definedits majorclinical manifestationsandrecognizedthe fact that it probablywasa hereditary disease. His description, however,wasmost completein that he definedall its clinical manifestations.In addition, by recognizing HANE in five successive generations of a family, there remainedno doubt that it washereditary. CLINICAL CHARACTERISTICS HANE is characterized by recurrent, acute, local circumscribed edemaof the skin or mucosa. The primary areas involvedby edemaare the extremities, face, larynx, and gastrointestinal tract (149-153). The edemais nonpitting and nonpruritic, and it is not painful exceptwith gastrointestinal tract involvement,in whichcase patients present with acute, crampyabdominalpain. Frequently,this pain is associated with nausea and vomiting, and less commonly with diarrhea. Themost serious, and potentially life-threatening manifestation of the disease is laryngeal edema.Prior to the institution of androgentherapy

Annual Reviews

Annu. Rev. Immunol. 1988.6:595-628. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

612

DAVIS

for HANE,death from laryngeal edema occurred commonly, but with wide variation from kindred to kindred (150, 151-155). Erythematous mottling, similar to erythema marginatum, is frequently observed but may occur with or without typical attacks (148, 150, 151, 156). Attacks usually last from 24 to 72 hr and are frequently preceded by trauma or emotional stress. They also occur in the absence of any identifiable precipitating event. The attacks usually begin during childhood (151, 152), although the severity frequently worsens during adolescence (151). Symptomstend diminish by the fifth or sixth decade of life (151). Manywomenreport increased frequency of attacks during menstrual periods (151,152, 157). Even more fascinating is the observation that the frequency of attacks decreases dramatically during pregnancy, particularly in the second and third trimesters (152). C1 INHlevels, however, reportedly are somewhat reduced during pregnancy (at least in normal women)(158-160), although this in itself is difficult to interpret since someof the enzymesregulated by C1 INH may also be diminished during pregnancy. C1 INH also is decreased during therapy with oral contraceptives (161). As mentioned previously, HANEoccurs in individuals with a genetically determined deficiency of C1 INH(100, 162), and it is inherited as an autosomal dominant trait (153). Donaldson& Evans (162) first discovered that these patients C1 INHdeficient, while in the preceding year, Landermanet al (100) had found that plasma from HANEpatients was deficient in the ability to inhibit kallikrein and PF/dil (factor XIIf). Because of the diminished levels of C1 INH, the C1 activation that occurs during an attack results in consumptionof its substrates, C2 and C4, and this results in diminished serum concentrations of both these proteins (154, 163, 164). C2 and levels may also be decreased between attacks, but they fail even further when symptoms develop. Activated C1 has been demonstrated in the plasma of patients during attacks of angioedema(163, 165). As was discussed previously, the precise mechanismleading to attacks has not been defined. Although contact system activation probably also occurs during attacks of angioedema, this has been more difficult to document, and a long delay followed Landerman’s(100) initial findings before any further evidence for contact system activation was obtained. Theoretically, such activation probably occurs since C1 INHis the primary inhibitor of both kallikrein and activated forms of factor XII (XIIa and XIIf) (81-84, l01106). In addition, trauma which can induce attacks of angioedema can also result in factor XII activation. In addition to activating kallikrein, factor XIIf activates C1 (166--170). Activated factor XII and kallikrein have also been reported to generate plasmin from plasminogen (171, 172). COMPLEMENTAND CONTACT SYSTEM ACTIVATION

Annual Reviews

Annu. Rev. Immunol. 1988.6:595-628. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

Cl INHIBITOR 613

Plasmin,in turn, can also activate C1and reportedlyreleases a kinin-like peptide fromC2(see further discussionbelow)(173, 174). Curdet al (175) showedthat a large amountof activated kallikrein waspresent in induced blister fluids of HANE patients. Additional evidencefor contact system activation wasprovided by the finding that three HANE patients, during attacks, had decreasedlevels of prekallikrein (a substrate of activated factor XII) and of high-molecular-weight kininogen(a substrate for kallikrein) (176). Zuraw& Curd(177) have identified a modified, inactive 94,000 form of C1 INHin the plasma of patients with HANE (and those with acquired C1INHdeficiency). This fragmentmayrepresent modified inhibitor released from the protease-C1 INHcomplexafter cleavage of the active center peptide bond;this has beenshownto occur in vitro with other protease-protease inhibitor complexes(178-183).Theseauthors and others have shownthe generation of a similar C1 INHfragment during contact systemactivation or with isolated plasmakallikrein, but not with complement activation or with purified activated Clr or Cls (9, 17, 18, 74, 82, 95, 120, 177, 184). Recently,Alsenzet al (185) also described inactive C1 INHfragment (Mr 96,000) in the plasma of patients with acquiredC 1 INHdeficiency. In contrast to the findings of Zuraw& Curd (177), however,this fragmentappears to be generated during complement systemactivation and specifically is a result of cleavage of C1INHby activated Cls. Incubationof ~25I-labeledC1INHin patients’ plasma(and in HANE patients’ plasma)resulted in generation of a fragmentidentical in size to the fragmentisolated frompatients’ plasma.Afragmentof the samesize resulted fromincubation of ~25I-C1INHwith isolated activated Cls, but not with activated Clr, plasmin, or kallikrein. Therelationship of these fragmentsand the apparentdiscrepancyin their generationremain to be resolved, but they are of obviousimportancein defining the pathophysiology of angioedema.Thus, although the initiating event for the induction of symptomsin HANE is not precisely defined, there is ample evidence for both complementand contact system activation during attacks. MEDIATION OFSYMPTOMS Determination of the mediator of symptomsin HANE has also proven to be a difficult problem.Largely becauseof the evidence for contact system activation, and evidence for generation of bradykinin in angioedemaplasma in vitro, it has been suggested that bradykinin is the primary mediator (175, 176, 186-188). Clinically, however,it seemsunlikely that symptoms are solely due to bradykinin. Subcutaneousinjection of bradykinin induces pain and the swelling is erythematous,while intravenous injection induces hypotension. Noneof these are characteristics of the swelling in HANE.

Annual Reviews

Annu. Rev. Immunol. 1988.6:595-628. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

614 DAVIS Donaldsonet al (189) identified and partially purified a peptide generated in vitro from HANE plasma. This peptide had kinin activity (smoothmusclecontracting), and it enhancedvascular permeability guineapig skin. Several lines of evidencesuggestedthat this factor was not bradykinin:it differedin size, electrophoreticmobility,isoelectric point and susceptibility to trypsin 089, 190). Additionalstudies suggestedthat C4andC2werenecessaryfor generationof this peptide, in that antibodies to C2and C4 inhibited appearance of the peptide in HANE serum(191). Anotherline of evidence whichsuggests that symptomsin angioedema depend upon complementsystem activation was the observation that intradermal injection of activated Cls inducedlbcal swelling that was nonpainfuland did not itch (192-194).Theactive enzymaticsite of Cls required for this activity (195). C2deficient individualsfailed to respond (193), whileone C3deficient patient respondednormally(196). C2deficient guinea pigs are also unresponsiveto intradermal injection of active Cls but do respond normallyif their circulating C2 levels are restored to approximately35 %of normalby intravenous infusion of C2(197). Thus, C2is required for Cls-inducedvascular permeability, although these data do not indicate from whichprotein the kinin is derived. Vascular permeability and smoothmusclecontracting activity is generatedin mixtures of Cls, C2, C4and plasmin(173), or in mixtures of Cls, C2, and plasmin without C4(174). In the latter example,a small peptide was shownto released from the earboxyterminus of C2b, and small synthetic peptides that mimicthe aminoacid sequencein this region inducedboth activities (174). Thesepeptides, however,had low specific activities in comparison with bradykinin. In another study, no kinin-like activity was generated, and no cleavage of C2or C2bcould be demonstratedwith plasmin(198). The mediation of symptomsin HANE obviously requires further study including both the completedefinition of the active peptides derivedfrom C2, and definitive structural analysis of the kinin isolated fromthe plasma of patients. Thepossibility that symptoms mayresult fromthe interaction of morethan one factor should probablyalso be considered. GENETICS HANE maybe divided into two types. In type I, which comprises 85%of affected kindred, a normal C1 INHprotein is present in plasma at reduced levels of from 5%to 30%of normal. Type II is characterized by normalto elevated antigenic levels of C1INHdue to the presenceof a dysfunctionalmutantprotein together with low levels of the normalprotein (199, 200). Fromdifferencesin electrophoreticmobility, the mutantproteins were shownto be heterogeneous(199). Thedysfunctional proteins are also heterogeneousin terms of function (184). Eachof eight different isolated dysfunctional proteins showeda different spectrumof

Annual Reviews

Annu. Rev. Immunol. 1988.6:595-628. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

C1 INHIBITOR

615

inhibitory activity with C 1 s, plasmakallikrein, activated Hageman factor, and plasmin. In addition, the patterns of complexformation with and cleavage by Cls and plasmin varied amongthe mutant proteins. From limited structural analysis of variant C1 INHproteins, data thus far available confirmthat the proteins are heterogeneous(165, 201-204). Since HANE is inherited as an autosomaldominantdisorder, affected individuals mustbe heterozygousfor the defect. The fact that levels of normalC1INHprotein in patients are muchless than 50%of normalled to the suggestionthat deficiency mightbe due to a regulator genedefect rather than a structural genedefect(205). In type II HANE, this is certainly not the case, since both normalandmutantprotein~are present in patients’ plasma(200, 202). TypeII patients, therefore, musthavea structural gene defect. In type II, like type I, the levels of normalCI INHare reduced below50%of normal. Lachmann& Rosensuggested that this reduction is due to the catabolic behavior of C1INH(206, 207), in whicha level 50%of normal cannot be maintained due to consumptionof C1 INHvia reaction with susceptible proteases. Analysisof the metabolismof C1INH in HANE patients and in normal individuals was consistent with this hypothesis(208). Thesynthesis of normalC 1 INHin patients wasapproximatelyhalf that of normalindividuals, as wouldbe expectedwith a heterozygousdeficiencystate. In addition, the fractional catabolic rate of the normalprotein was elevated in patients to a degree consistent with the reduction in C1INHlevels below50%of normal(208). Study of the in vitro synthesis of C1INHby cultured monocytesfrom individuals with type I HANE was also consistent with the aboveobservation (45). Analysis of biosynthetically labeled C1 INHby immunoprecipitation and SDS-polyacrylamide gel electrophoresis revealed intracellular C1INHlevels that were approximately50%of normal, as were C1 INHmRNA levels. C1 INHlevels in the supernatants of cultured monocyteswere only 20%of normal levels and approximatedthe quantities foundin the patients’ serum. Pulse chase experimentsrevealed no evidencefor a secretory defect. Thesedata suggestthat CI INHin patient monocytecultures is consumed during or after secretion and is therefore compatiblewith the above suggestion. Twoother observations further showthat HANE (both type I and type II) is due to mutationsin the structural gene for C1INH.Atype I HANE patient was identified in whoma normal appearing C1 INH mRNA was detected at concentrations about 50%of normal together with an abnormal C1 INH mRNA that was 0.2 kb smaller than normal (45). Analysis of the patients’ serumand cultured monocytesrevealed no indication that translation of this mRNA occurred. Theonly available family membersof this patient were two siblings, one of whomhas HANE. The

Annual Reviews

Annu. Rev. Immunol. 1988.6:595-628. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

616

DAVIS

sibling with HANEhad the same abnormality, while the normal sibling had only a normal appearing message. This suggests that the abnormality is genetically determined and is related to the presence of HANE. Restriction fragment length polymorphism (RFLP) of the C1 INHgene recently has been observed (209, 210). In every instance, the polymorphismcosegregated with the disease (with low C1 INHlevels), thus further indicating that a defective structural gene is responsible for the disease. Several other informative findings were derived from these studies. Stoppa-Lyonnet et al (209) found RFLPsin 4 of 7 kindred analyzed, while Cicardi et al (210) found RFLPsin only 3 of 29 kindred. Most mutations resulting in C1 INH deficiency do not produce RFLPsand therefore probably are due to relatively "minor" defects; manymay result from single base change mutations. In somefamilies (210), the presence of multiple restriction site markers are consistent with structural rearrangements. RFLPanalysis also showsthat type I HANE, like type II, is genetically heterogeneous.All the 7 kindred with RFLPs(209, 210), show different polymorphisms. However, Southern blot analysis of these families with smaller eDNA probes suggests the possibility that there maybe specific regions within the C1 INHgene that are more susceptible to mutation. A further indication of the genetic heterogeneity of HANE was the observation that three families with type ! C1 INHdeficiency (none of whomrevealed RFLPs) had elevated levels of an apparently nonfunctional mRNA that was of normal size (211). The heterogeneity of the defects and the possibility of hypermutable regions within the gene will ultimately be defined by analysis of genomic (and cDNA)clones from multiple individuals with both type I and type II HANE. THERAPY Long-term preventive therapy in HANEis now based largely on the use of attenuated androgens. In 1960, Spaulding first showed that methyltestosterone was effective in preventing attacks (212). It interesting that the theoretic basis for using testosterone was that it might interfere with the action of histamine. Although it was later shownthat patients with HANEdo have histaminuria, antihistaminic agents have no effect on symptomsin the disease (213, 214). Subsequently, after it was shown that HANEwas due to C1 INH deficiency, several groups confirmed the clinical observations and showed that C1 INHand C4 levels increased during therapy with androgens (215-217). In 1976, Gelfand al (218) showed that the attenuated androgen danazol also prevented attacks of edema and resulted in increased serum levels of C1 INH, thus avoiding manyof the undesirable side effects of testosterone. Numerous studies have since confirmed the efficacy of impeded androgens (danazol and, more recently, stanazolol), and have shownthat 17 alpha-alkylation

Annual Reviews C1 INHIBITOR

617

Annu. Rev. Immunol. 1988.6:595-628. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

of the androgenis necessaryfor activity, that C4and C2levels rise along with C1INHlevels, andthat it is not necessaryto attain levels withinthe normalrange for therapeutic efficacy (219-223). The mechanism by which androgensexert their effect on C1 INHlevels remains to be defined, althoughpresumablysynthesis by the liver is enhanced.In addition to its implications for therapy, study of the effect of attenuated androgenson C1 INHsynthesis mayprovide a useful modelfor the study of hormone inducible gene expressionand hormone-receptorinteractions. The Syndrome of Acquired C1 INH Deficiency AcquiredC1INHdeficiency is characterized by the adult onset of angioedemain an individual with no evidencefor inheritance of the disorder. Although less commonthan HANE,numerousreports have appeared in the literature since the first descriptionin 1969(185, 224-239).Analysis the complementsystem in these patients reveals decreased or absent C1 INH, Clq, C1, C4, and C2. The low levels of Clq and C1 provide an important characteristic distinguishing them from HANE. The synthesis of C1INHin acquired C1 INHdeficiency is similar to that in normal individuals, while its catabolismis markedlyelevated comparedwith both normals and HANE patients (240). The fractional catabolic rate of Clq was also elevated. Most reported cases of acquired C1 INHdeficiency havebeen associated with benignor malignantB-cell lymphoproliferative disorders, such as B-cell lymphosarcoma, chronic lymphocyticleukemia, macroglobulinemia,multiple myelomaand essential cryoglobulinemia. Several reports describe acquired C1INHdeficiency in association with other diseases or its occurrencewithoutassociateddisease (185, 230, 231, 235, 239, 241,242). Someindications suggest these maybe, in fact, two separate disorders (185, 237, 239, 241,242). Four patients with C1 INH deficiencyassociated with B-cell disorders werefoundto havecirculating anti-idiotypic antibodies directed toward the monoclonalimmunoglobulin expressedon the surface of their B-cells or in the cytoplasmof their bone marrowcells (237). Theanti-idiotypic antibodies from the two patients with circulating M-componentsreacted with these M-components.In addition, an increased quantity of Clq was present on the surface of the patients’ B-cells. Thesedata suggest that immunecomplexesconsisting of the idiotype of monoclonalimmunoglobulinand the anti-idiotypic antibodies bind and consumeC1and, ultimately, C1 INH,leading to the symptomsof angioedema(237). The second type of acquired C1 INH deficiencyhas beenreported in five individuals(185, 239, 241,242)and characterized by the presence of autoantibodiesto the C 1 INHmolecule. Noneof these five patients had detectable associated lymphoproliferative disorders; they wereotherwiseclinically indistinguishablefromother pa-

Annual Reviews

Annu. Rev. Immunol. 1988.6:595-628. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

618 DAVIS tients with acquireddeficiency. In three instances the autoantibodieswere of the IgG class; in the other two, they wereIgA. Theseautoantibodies, whichare probablydirected towardthe C1INHactive site, inactivate C1 INHfunction by preventing its binding to activated Cls. This allows C1 activation (and presumablyother susceptible enzymes),as in HANE, with ultimate developmentof symptomsof angioedema.It, therefore, appears that acquired C1INHdeficiency mayresult from two different autoantibody-mediatedmechanisms.In one, the autoantibody is directed toward and inactivates C1INH.In the other, autoanti-idiotypic antibodies complex with monoclonalimmunoglobulin (either circulating in plasmaor on the surface of B-lymphocytes),efficiently activate C1, and thus consume C1 INH. Literature Cited 1. Lepow,I. H., Ratnoff, O. D., Rosen, F. S., Pillemer, L. 1956.Observations on a proesterase associated with partially purified first component of complement (C1). Proc. Soc. Exp. BioL Med. 92:32-37 2. Ratnoff, O. D., Lepow,I. H. 1957. Someproperties of an esterase derived from preparations of the first component of complement.J. Exp. Med. 106:327-43 3. Lepow,I. H., Ratnoff, O. D., Levy,L. R. 1958. Studies on the activation of a proesterase associatedwith partially purified first componentof human complement.J. Exp. Med. 107: 45174 4. Levy,L. R., Lepow,I. H. 1959. Assay and properties of seruminhibitor of C’I esterase. Proc.Soc. Exp. Biol. Med. 101:608-11 5. Pensky,J., Levy,L. R., Lepow,I. H. 1961. Partial purification of a serum inhibitor ofC’lesterase. J. Biol. Chem. 236:1674-79 6. Schultze, H. E., Heide, K., Haupt, H. 1962. Uber ein bisher inbekanntes saures alpha 2 glykoprotein. Naturwissenschaften49:133-34 7. Pensky, J., Schwick, H. G. 1967. Humanserum inhibitor of C’l-esterase: identity with alpha 2-t~euraminoglycoprotein. Science 163:698-99 8. Haupt, H., Heimburger,N., Kranz,T., Schwick,H. G. 1970. Ein beitrag zur isolierung und characterisierung des Cl-inaktivators aus humanplasma. Eur. J. Biochem.17:254~51 9. Harpel, P. C., Cooper, N. R. 1975. Studies on human plasma Cl-inac-

tivator-enzymeinteractions. I. Mechanismsof interaction withC1 s, plasmin andtrypsin. J. Clin. Invest. 55:593-604 10. Reboul,A., Arlaud,G. J., Sire, R. B., Colomb,M.G. 1977. A simplified procedurefor the purification of Cl-inactivator from humanplasma. Interaction with complement subcomponents Clr and Cls. FEBSLett. 79: 45-50 11. Harrison, R. A. 1983. HumanC1inhibitor: improvedisolation and preliminary structural characterization. Biochemistry 22:5001-7 12. Nilsson, T., Wiman,B. 1982. Purification and characterization of human Cl-esterase inhibitor. Biochim. Biophys. Acta 705:271-76 13. Odermatt, E., Berger, H., Sano, Y. 1981. Size and shape of humanCIinhibitor. FEBSLett. 131:283-85 14. Minta,J. O. 1981. Therole of sialic acid in the functional activity andthe hepatic clearance of C 1-INH.J. ImmunoL 126:245-49 15. Prandini, M. H., Reboul, A., Colomb, M. G. 1986. Biosynthesis of complementC 1 inhibitor by HepG2 cells. Reactivity of different glycosylatedformsof the inhibitor with Cls. Biochem.J. 237:93-98 16. Bock,S. C., Skriver, K., Nielsen, E., Thogersen, H.-C., Wiman,B., Donaldson, V. H., Eddy,R. L., Marrinan, J., Radziejewska, E., Huber, R., Shows, T. B., Magnusson,S. 1986. HumanC1 inhibitor: primary structure, cDNAcloning, and chromosomal localization. Biochemistry 25: 42924301

Annual Reviews

Annu. Rev. Immunol. 1988.6:595-628. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

C1 INHIBITOR 17. Salvesen,G. S., Catanese,J. J., Kress, L. F., Travis,J. 1985.Primarystructure of the reactive site of humanCl-inhibitor. J. Biol. Chem.260:2432-36 18. Nilsson, T., Sjoholm, I., Wiman,B. 1983. Structural andcircular dicroism studies on the interaction between humanCl-esterase inhibitor and Cls. Biochem.J. 213:617-24 19. Hunt, L. T., Dayhoff, M. O. 1980. A surprising newprotein superfamily containing ovalbumin, antithrombin III, and alpha-l-proteinase inhibitor. Biochem. Biophys. Res. Commun. 95: 864-71 20. Morii, M., Travis, J. 1983. Amino acid sequenceat the reactive site of humanalpha-1-antichymotrypsin. J. Biol. Chem. 258:12749-52 21. Hill, R. E., Shaw,P. H., Boyd,P. A., Baumann,H., Hastie, N. D. 1984. Plasmaprotease inhibitors: divergence within the reactive centre region. Nature311: 175-77 22. Ragg, H. 1986. A new memberof the plasmaprotease inhibitor genefamily. Nucleic Acids Res. 14:1073-88 23. Holmes,W.E., Nelles, L., Lijnen, H. R., Collen, D. 1987.Primarystructure of humanalpha-2-antiplasmin,a serine protease inhibitor (serpin). J. Biol. Chem. 262:1659q54 24. Minta,J. O., Aziz, E. 1980.Analysisof the reactive site peptide bondin C1inhibitor by chemicalmodificationof tyrosyl, lysyl, andarginyl residues:the essential role of lysyl residues in the functional activity of CI-INH.J. Immunol. 126:250-55 25. Sim, R. B., Reboul, A. 1981. Preparation and properties of humanC1 inhibitor. MethodsEnzymol.80:43-54 26. Ishizaki, E., Mori, Y., Koyama,J. 1977. Purification and someproperties of rabbit C1inactivator. J. Biochem. (Tokyo) 82:1155-60 27. Loos, M., Opferkuch, W., Ringelmann, R. 1971. Studien uber den C1inaktivator des Meerschweinchenkomplements: Messmethode, reinigungund charakterisierung des proteins. Z. Med.Microbiol.Immunol.156: 194-207 28. Loos, M. 1983. The classical complement pathway: mechanism of activation of the first componentby antigen-antibodycomplexes.Pro#. A1lergy 30:135-92 29. Gigli, I.,Austen, K. F. 1971.Phylogeny and function of the complement system. Ann. Rev. Microbiol. 25: 30932 30. Davis,A. E. III, Whitehead, A. S., Har-

619

rison, R. A., Dauphinais,A., Bruns,G. A. P., Cicardi, M., Rosen,F. S. 1986. Humaninhibitor of the first component of complement,CI: characterization of eDNAclones and localization of the gene to chromosome 11. Proc. Natl. Acad. Sci. USA83: 316165 C., Bourgarel,P., 31. Tosi, M., Duponchel, Colomb,M., Meo,T. 1986. Molecular cloning of human C1 inhibitor: sequence homologies with alpha-1antitrypsin and other membersof the serpins superfamily. Gene42:265-72 32. Que,B. G., Petra, P. H. 1986.Isolation and analysis of a. cDNAcoding for humanC1inhibitor. Biochem.Biophys. Res. Commun.137:620-25 33. Can’ell, R. W., Boswell,D. R. 1986. Serpins: the superfamilyof serine proteinaseinhibitors. In ProteinaseInhibitots, ed. A. Barrett, G. Salvesen,pp. 403-20. Amsterdam:Elsevier 34. Purrello, M., Alhadeff, B., Esposito, D., Whittington,E., Daniel, A., Buckton, K. E., Siniscalco, M. 1985. The subregional assignment of the human locus for alpha-l-antitrypsin (PI) band 14q 32.1 suggests uneven distribution of crossingovereventsin the distal third of autosome14q. Cyto#enet. Cell Genet.40:725(Abstr.) 35. Rabin, M., Watson, M., Kidd, V., Woo,S. L., Breg, W. R., Ruddle, F. H. 1986.Regionallocation of alpha-1antichymotrypsin and alpha-l-antitrypsin genes on humanchromosome 14. SomaticCell Mol. Genet.12: 20914 36. Loebermann, H., Tokuoka, R., Diesenhofer, J., Huber, R. 1984. Humanalpha-l-proteinase inhibitor. Crystalstructure analysisof twocrystal modifications, molecular model and preliminaryanalysisof the implications for function. J. Mol.Biol. 177:531-57 37. ,’ttlas of Protein Sequenceand Structure. 1985. Natl. Biomed.Res. Found., Washington, DC 38. Heilig, R., Muraskowsky, R., Kloepfer, C., Mandel,J. L. 1982. The ovalbumin gene family: complete sequenceand structure of the Y gene. Nucleic Acids Res. 10:4363-82 39. Ny, T., Sawdey,M., Lawrence, D., Millan, J. L., Loscutoff, D. J. 1986. Cloning and sequence of a cDNA coding for the humanbeta-migrating endothelial-cell-typeplasminogen activator inhibitor. Proc.Natl. Acad.Sci. USA83:6776-80 40. Hejgaard, J., Rasmussen, S. K., Brandt, A., Svendsen, I. 1985. Se-

Annual Reviews

Annu. Rev. Immunol. 1988.6:595-628. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

620

DAVIS

quencehomologybetweenbarley endospermprotein Z and protcase inhibitors the 1-antitrypsin family. FEBS Lett. of180:89-94 41. Pickup,D. J., Ink, B. S., Hu,W.,Ray, C. A., Joklik, W.K. 1986. Hemorrhage in lesions caused by cowpoxvirus is inducedbya viral proteinthat is related to plasmaprotein inhibitors of serinc protcascs. Proc. Natl. Acad.Sci. USA 83:7698-7702 42. Cox, D. W., Markovic,V. D., Tcshima, I. E. 1982. Genesfor immunoglobulin heavychains and for l-antitrypsin are localized to specific regionsof chromosome14q. Nature 297:428-30 43. Kao,F. T., Morse,H. G., Law,M. L., Lidsky,A., Chandra,T., Woo,S. L. C. 1983.1-antitrypsindeficiencydetection bydirect analysisof the mutationin the gene. Nature 304:230-34 44. Bock,S. C., Harris, J. F., Balazs, I., Trent, J. M. 1985. Assignmentof the humanantithrombin III structural gene to chromosomelq 23-25. Cyto#enet. Cell Genet.39:67~i9 45. Cicardi,M., Igarashi, T., Rosen,F. S., Davis,A. E. III. 1987.Molecularbasis for the deficiency of complement1 inhibitor in type 1 hereditaryangioneurotic edema.J. Clin. Invest. 79: 698702 46. Johnson,A. M., Alper, C. A., Rosen, F. S., Craig, J. M.1971.C1inhibitor: evidence for decreased hepatic synthesis in hereditary angioneurotic edema. Science 173:553-54 47, Gitlin, D., Biasucci, A. 1969. Developmentof G, A, M, IC/1A, C’l esterase inhibitor, ceruloplasmin, transferrin, hemopexin, haptoglobin, fibrinogen, plasminogen,1-antitrypsin, orosomucoid, lipoprotein, 2-macroglobulin, and prealbuminin the human conceptus.J. Clin. Invest. 48:1433-46 48. Morris, K. M., Aden,D. P., Knowles, B. B., Colten, H. R. 1982. Complement biosynthesis by human hepatoma derivedcell line HepG2. J. Clin. Invest. 70:906-13 49. Perlmutter, D. H., Cole, F. S., Kilbridge, P., Rossing,T. H., Colten, H. R. 1985. Expressionof the 1-proteinase inhibitor gene in humanmonoeytes Sci. and macrophages. Proc. Natl. Acad. USA82:795-99 50. Yeung-Laiwah, A. C., Jones, L., Hamilton, A. O., Whaley,K. 1985. Complement subcomponentC1 inhibitor synthesis by humanmonocytes. Biochem. J. 226:199-205 51. Bensa, J. C., Reboul, A., Colomb,M. G. 1983.Biosynthesisin vitro of corn-

plement subcomponentClq, Cls, and C1inhibitor by resting and stimulated humanmonocytes. Biochem. J. 216: 385-92 52. Randazzo,B. P., Dattwyler, R. J., Kaplan, A. P., Ghebrehiwet,B. 1985. Synthesis of C1 inhibitor (CI-INA) aJ. human monocyte-like cell line, U937. Immunol. 135:1313-19 53. Endresen, G. K. M. 1980. Immunological studies of plasma protease inhibitors associated with human blood platelets. Thromb.Res. 19: 15763 54. Schmaier, A. H., Smith, P. M., Colman,R. W.1985.Platelet C1inhibitor. Asecreted J. Clin. lnvest. alpha-granule 75:242-50 protein. 55. Ratnoff,O. D., Pensky,J., Ogston,D., Naff, G.B. 1969.Theinhibitionof plasrain, plasmakallikrein, plasmapermeability factor, and the Cl’r subcomponentof the first componentof complement by serum CI’ esterase inhibitor. J. Exp. Med.129:315-31 56. Gigli, I., Mason,J. W., Colman,R. W., Austen,K. F. 1970. Interaction of plasma kallikrein withthe C1 inhibitor. J. Immunol. 104:574-81 57. Forbes,C. D., Pensky,J., Ratnoff, O. D. 1970. Inhibition of activated Hageman factor and activated plasma thromboplastinantecedent by purified C1inactivator. J. Lab. Clin. Med.76: 809-15 58. Schreiber, A. D., Kaplan, A. P., Austen, K. F. 1973. Inhibition by C1INHof Hageman factor fragment activation of coagulation,fibrinolysis, and kinin generation. J. Clin. Invest. 52: 1402-9 59. Sim,R. B., Reboul,A., Arlaud,G. J., Villiers, C. L., Colomb,M. (3. 1979. Interaction of 1:SI-labeled complement componentsClr and Cls with protease inhibitors in plasma. FEBSLett. 97: 111-15 60. Ziccardi, R. J. 1981. Activationof the early components of the classical complement pathwayunder physiological conditions. J. Immunol.126:1768-73 61. Brower,M.S., Harpel,P. C. 1982. Proteolytic cleavageandinactivation of 2plasmininhibitor and C1 inactivator by humanpolymorphonuclear leukocyte elastase. J. Biol. Chem.257:9849-54 62. Kress, L. F., Catanese, J., Hirayama, T. 1983.Analysisof the effects of snake venomproteinases on the activity of humanplasmaC1 esterase inhibitor, 1antichymotrypsin,and 2-antiplasmin. Biochim. Biophys. Acta 745:113-20 63. Catanese,J., Kress, L. F. 1984. Enzy-

Annual Reviews

Annu. Rev. Immunol. 1988.6:595-628. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

CI INHIBITOR 62I matic inactivation of humanplasma Cl-inhibitor and 1-antichymotrypsin by Pscudomonas aeruginosa proteinase and elastase. Biochim. Biophys. Acta 789:37-43 64. Laskowski,M. Jr., Kato, L 1980. Protein inhibitors of proteinases.Ann.Rev. Biochem. 49:593-626 65. Travis, J., Salvesen,G. S. 1983.Human plasmaproteinascinhibitors. Ann.Rev. Biochem.52:655-709 66. Schechter,I., Berger,A. 1967. Onthe active site of protcascs.I. Papain.Biochem. Biophys. Res. Commun. 27: 15762 67. Ziccardi, R. J., Cooper,N. R. 1976. Activationof Clr by proteolytic cleavage. J. Immunol.116:504-9 68. Nagaki,K., Iida, K., Inai, S. 1974.The inactivator of the first componentof human complement (C1 INA). The complexformation with the activated first componentof humancomplement (C1) or with its subcomponents.Int. Arch. Allergy Appl. Immunol.46: 93548 69. Arlaud, G., Reboul, A., Sire, R., Colomb,M. 1979. Interaction of C1inhibitor with the Clr and Cls subcomponents in humanC1. Biochim. Biophys. Acta 576:151-62 70. Haines,A. L., Lcpow,I. H. 1964.Studies on humanC’l-esterase. I. Purification and enzymaticproperties. J. Immunol.92:456-67 71. Bing, D. H. 1969. Natureof the active site of a subunit of the first component of humancomplement.Biochemistry8: 4503-10 72. Sire, R. B., Arlaud,G. J., Colomb,M. (3. 1979. C1 inhibitor dependentdissociation of humancomplementcomponent C1 bound to immune complexes. Biochem.J. 179:449-57 73. Weiss, V., Engel, J. 1983. Heparinstimulated modificationof Cl-inhibitor by subcomponent Cls of human complement.Hoppe-Seyler’sZ. PhysioL Chem.364:295-301 74. Sire, R., Arlaud,G., Colomb,M. 1980. Kinetics of reaction of humanCIinhibitor with the humancomplement systemproteases Clr and Cls. Biochim. Biophys. Acta 612:433-49 75. Lennick, M., Brew,S. A, Ingham,K. C. 1986.Kinetics of interaction of CI inhibitor with complementCls. Biochemistry 25:3890-98 76. Rent, R., Myhrman, R., Fiedel, B. A., Gewurz,H. 1976. Potentiation of C1esterase inhibitor activity by heparin. Clin. Exp. Immunol.23:246-71 77. Caughman,G. B., Boackle, R. J.,

Vesely, J. 1982. A postulated mechanismfor heparin’s potentiation of CI inhibitor function. Mol. lmmunol.19: 287-95 78. Nagaki,K., Inai, S. 1976. Inactivator of the first componentof humancomplement (CIlNA). Enhancement CIINAactivity against Cls by acidic mucopolysaccharides.Int. Arch. Allergy Appl. lmmunol.50:172-80 79. Raepple,E., Hill, H. V., Loos,M.1976. Modeof interaction of different polyanions with the first (C1,C1), the second(C2) and the fourth (C4) ponent of complement.I. Effect on fluid phase C1 and on C1 boundto EA or to EAC4.Immunochemistry 13: 25155 80. Loos, M., Volanakis,J. E., Stroud, R. M. 1976. Modeof interaction of different polyanions with the first (C1,C1)the second(C2) and the fourth (C4) componentof complement.II. Effect of polyanionson the bindingof C2 to EAC4b.Immunochemistry 13: 257-61 81. Schapira,M., Scott, C. F., Colman,R. W.1981. Protection of humanplasma kallikrein from inactivation by C1inhibitor andother proteaseinhibitors. The role of high molecular weight kininogen. Biochemistry20:2738-43 82. van der Graaf, F., Koedam,J. A., Giffin, J. H., Bouma, B. N.1983.Interaction of humanplasmakallikrein and its light chain withC1 inhibitor. Biochemistry 22:4860-66 83. de Agostini,A., Lijnen, H. R., Pixley, R. A., Colman,R. W., Schapira, M. 1984.Inactivationof factor XII active fragment in normal plasma. Predominant role of CI-inhibitor. J. Clin. Invest. 73:1542-49 84. Pixley, R. A., Schapira, M., Colman, R. W.1985. The regulation of human factor XIIa by plasma proteinase inhibitors. J. Biol. Chem.260:1723-29 85. Colman, R. W., Waehtfogel, Y. T., Kucich, U., Weinbaum,G., Hahn,S., Scott,C. F., de Agostini,A., Burger,B., Sehapira, M.1985. Effect of cleavage of the heavy chain of humanplasma kallikrein on its functionalproperties. Blood 65:311-18 86. Schapira, M., de Agostini, A., Sehifferli, J. A., Colman,R. W.1985. Biochemistry and pathophysiologyof humanC1 inhibitor: current issues. Complement2:111-26 87. Scott, C. F., Schapira, M., James,H. L., Cohen,A. B., Colman,R. W.1982. Inactivation of factor XIa by plasma protease inhibitors. Predominantrole

Annual Reviews

Annu. Rev. Immunol. 1988.6:595-628. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

622

DAVIS

of 1-protease inhibitor and protective effect of high molecularweightkininogen. J. Clin. Invest. 69:844-52 88. Schapira,M., Scott, C. F., James,A., Silver, L. D., Kueppers,F., James,J. L., Colman,R. W. 1982. High molecular weightkininogenor its light chain protects humanplasmakallikrein from inactivation by plasmaprotease inhibitots. Biochemistry21:567-72 89. Silverberg, M., Longo,J., Kaplan,A. P. 1986. Studyof the effect of high molecular weight kininogen upon the fluid-phase inactivation of kallikrein by Cl-inhibitor. J. Biol. Chem.261: 14965~58 90. Ziecardi, R. J., Cooper, N. R. 1978. Modulationof the antigenicity of Clr and Cls inactivator. J. ImmunoL 121: 2148-52 91. Ziccardi, R. J., Cooper,N. R. 1978. Demonstration and quantitation of activation of the first componentof complementin humanserum. J. Exp. Med. 147:385-95 92. Yan, D., Gu, X., Wang,D., Yang,S. 1981. Studies on immunopathogenesis in epidemichemorrhagicfever: sequential observationson activation of the first complementcomponentin sera from patients with epidemic hemorrhagic fever. J. Immunol.127:1064-67 93. Cooper, N. R., Nemerow, G. R., Mayes,J. T. 1983. Methodsto detect and quantitate complement activation. Sprin#er Semin.Immunopathol. 6: 195212 94. Ziccardi, R. J., Cooper, N. R. 1980. Development of an immunochemical test to assess C1inactivator functionin humanserumand its use for the diagnosis of hereditary angioedema.Clin. Immunol. Immunopathol.15:465-71 95. de Agostini, A., Schapira, M., Wachtfogel, Y. T., Colman,R, W., Carrell, S. 1985. Humanplasma kallikrein and Cl-inhibitor form a complex possessingan epitopethat is not detectable on the parent molecules: demonstration using a monoclonalantibody. Proc. Natl. Acad, Sci. USA82: 519093 96. Collen, D. 1974. Emergencein plasma during activation of the coagulationor fibrinolytic systemsof neoantigensassociated with the complexesof thrombin or plasmin with their inhibitors. Thromb.Res. 5:777-79 97. Lau, H. K., Rosenberg, R. D. 1980. Theisolation andcharacterizationof a specific antibody population directed against the thrombin-antithrombin complex.J. Biol. Chem.255:5885-93

98. McDutiie,F. C., Peterson,J. M., Clark, G., Mann, K. G. 1981. Antigenic changes produced by complex formation between thrombin and antithrombin III. J. lmmunol.127: 23999. Wallgren,P., Nordling, K., Bjork, I. 1981. Immunologicalevidence for a proteolytic cleavageat the active site of antithrombin in the mechanism of inhibition of coagulation serine proteases. Eur. J. Biochem.116: 49396 100. Landerman,N. S., Webster, M. E., Becker,E. L., Ratcliffe, H. E. 1962. Hereditary angioneurotic edema.II. Deficiencyof inhibitor for serumglobulin permeabilityfactor and/or plasma kallikrein. J. Alleryy33:330-41 101. McConnell,D. J. 1972. Inhibitors of kallikrein in humanplasma. J. Clin. lnvest. 51:1611-23 102. Gallimore, M. J., Amundsen, E., Larsbraaten, M., Lyngaas,K., Fareid, E. 1979. Studies on plasmainhibitors of plasmakallikrein using chromogenic peptide substrate assays. Thromb.Res. 16:695-703 103. Schapira,M., Scott, C. F., Colman,R. W.1982. Contribution of plasma protease inhibitors to the inactivation of kallikrein in plasma.J. Clin. Invest. 69: 46248 104. van der Graaf, F., Koedam,J. A., Bouma,B. N. 1983. Inactivation of kallikrein in humanplasma. J. Clin. Invest. 71:149-58 105. Lewin,M. F., Kaplan, A. P., Harpel, P. C. t983. Studies of C1inactivatorplasmakallikrein complexes in purified systemsand in plasma.Quantitationby an enzyme-linked differential antibody immunosorbentassay. J. Biol. Chem. 258:6415-21 106. Harpel, P. C., Lewin, M. F., Kaplan, A. P. 1985. Distribution of plasma kallikrein betweenC1inactivator and 2-macroglobulinin plasmautilizing a newassay for 2-macroglobulin-kallikrein complexes.J. Biol. Chem.260: 4257~3 107. Aoki, N., Moroi, M., Matsuda, M., Tachiya, K. 1977. The behavior of 2plasmin in fibrinolytic states. J. Clin.inhibitor Invest. 60:361-69 108. Harpel, P. C. 1981.2-plasmininhibitor and 2-macroglobulin-plasmin complexes in plasma, Quantitation by an enzyme-linkeddifferential antibody immunosorbent assay. J. Clin. lnvest. 68:46-55 109. Ziccardi, R. J., Cooper,N. R. 1977. The subunit composition and sedi-

Annual Reviews

Annu. Rev. Immunol. 1988.6:595-628. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

el

INHIBITOR 623

mentationproperties of humanC1. J. holm, A. G. 1976. C1 su~omponent complexesin normal and patholoImmunol, 118:2047-52 gical sera studied by crossed immuno110. Tschopp,J., Villiger, W., Fuchs,H., electrophoresis. Acta Pathol. MicroKilchherr,E., Engel,J. 1980.Assembly of subcomponents Clr and Cls of first biol. Stand. 84:455-64 componentof complement: electron 122. Laurell, A. B,, Johnson,U., Martensmicroscopicandultracentrifugal studson, U., Sjoholm,A. G. 1978. Formaies. Proc. Natl. Acad. Sci. USA77: tion of complexescomposedof Clr, 7014-18 C1s and C1inactivator in humanserum V. N., Poon, 111. Siegel, R. C., Schumaker, on activation of C1. Acta Pathol. P. K. 1981. Stoichiometry and sediMierobiol. Seand. 86C: 299-306 mentation properties of the complex 123. Ziccardi, R. J., Cooper,N. R. 1979. formedbetweenthe Clq and Clr2Cls2 Active disassemblyof the first comsubcomponents of the first component plement component,C1, by C1 inacof complement.J. ImmunoL 127: 2447tivator. J. Immunol,123:788-92 52 124. Dickler, H. B., Kunkel,H. G. 1972. 112. Ziccardi, R. J. 1983. Nature of the Interaction of aggregated -globulin with B lymphocytes.J. Exp. Med.136: metal ion requirementfor assemblyand function of the first componentof 191-96 human complement. J. Biol. Chem. 125. Sobel, A. T., Bokisch, V. A. 1975. 258:6187-92 Receptorfor C lq on peripheral human 113. Hughes-Jones,N. C., Gorick, D. B. lymphocytes and human lympho1982.Thebindingandactivation of the blastoid cells. Fed. Proe. 34:965 C1 r-C1 s subunitof the first component (Abstr.) of humancomplement.Mol. Immunol. 126. Suba, E. A.,Csakao,G. 1976. Clq (C1) receptor on humanplatelets: inhibition 19:1105-12 114. Siegel, R. C., Schumaker,V. N. 1983. of collagen-induced platelet aggreMeasurement of the association congation by Clq (C1) molecules.J. Immustants of the complexesformedbetween nol. 117:304-9 intact Clqor pepsin-treatedClqstalks 127. Wautier,J. L., Souchon,H., Reid, K. andthe unactivatedor activated Clr2s2 B. M., Peltier, A. P., Caen,J. P. 1977. tetramers. Mol. Immunol.20:53-66 Studies on the modeof reaction of the 115. Ziccardi, R. J. 1984. The role of first componentof complementwith immune complexesin the activation of platelets: interaction betweenthe colthe first componentof humancomlagen-likeportionof C1q andplatelets. plement. J. Immunol.132:283-88 Immunochemistry14:763-66 116. Sakai, K., Stroud, R. M. 1973. Puri128. Gabay, Y., Perlmann, H., Perlmann, fication, molecularproperties, andactiP., Sobel, A. T. 1979. A rosette assay vation of C1proesterase, Cls. J. Immufor the determinationof Clq receptorbearingcells. Eur. J. Immunol.9: 797nol. 110:1010-20 117. Valet, G., Cooper, N. R. 1974. Iso801 lation and characterizationof the pro- 129. Shadworth,M. F,, Cunningham, P. H., enzymeform of the Cls subunit of the Andrew,P. S. 1979. Thedemonstration first componentof complement.J. of complement C l q and Fc-IgGreceplmmunol. 112:339-50 tors on the surface of humanendo118, Ziccardi, R. J. 1982.Spontaneous actithelial ceils. Fed.Proc.38:1075 (Abstr.) vation of the first component of human 130. Tenner, A. J., Cooper, N. R. 1980. complement(CI) by an intramolecular Analysis of receptor-mediated Clq autocatalytic mechanism.J. Immunol. binding to humanperipheral blood 128: 2500~ mononuelearcells. J. Immunol.125: 119. Ziccardi, R. J. 1982. A newrole for 1658-64 Cl-inhibitor in homeostasis:control of 131. Tenner, A. J., Cooper, N. R. 1981. activation of the first componentof Identificationof types of cefis in human humancomplement.J. lmmunol.128: peripheral blood that bind Clq. J. 2505-8 Immunol. 126:1174-79 120. Chesne,S., Villiers, C. L., Arlaud,G. 132. Tenner, A. J., Cooper, N. R. 1982. J., LaCroix, M. G., Colomb, M. G. Stimulation of a humanpolymorpho1982. Fluid-phase interaction of C1 nuclear leukocyte oxidative response inhibitor (C1 INH)and the subcomby the Clqsubunit of the first compleponentsClr and Cls of the first comment component. J. Immunol. 128: ponent of complement,CI. Biochem. 2547-52 J. 201:61-70 133. Ghebrehiwet, B. 1984. Identification of 121. Laurell, A. B., Martensson,U., Sjothe Raji cell membrane-derivedClq

Annual Reviews

Annu. Rev. Immunol. 1988.6:595-628. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

624

DAVIS

inhibitor as a receptor for humanClq. Y., Fujii, S. 1984. Detectionof interJ. Exp. Med. 160:1375-89 mediaryClr with completeactive site, 134. Peerschke,E. I., Ghebrehiwet, B. 1987. using a synthetic proteinaseinhibitor. Human blood platelets possess specific FEBSLett. 172:159~i2 bindingsites for C1q.J. Immunol.138: 147. Ziccardi, R. J. 1983. The first com1537-41 ponent of human complement(CI): 135. Ghebrehiwet,B. 1986. Production and activation and control. SpringerSemin. characterization of a murine monolrnmunopathoL6:213-30 clonal IgM antibody to human Clq 148. Osier, W.1888. Hereditary anglo-neureceptor (C 1 qR). J. ImmunoL 137:618rotic oedema.Am.J. Med.Sci. 95: 36224 67 136. Doekes, G., VanEs, L. A., Daha, M. 149. Quincke, H. 1882. Uber akutes umR. 1983.C1inactivator: its efficiency schriebenes H autodem. Monatsschr. as a regulator of classical complement Parkt. Dermatol.1:129-31 pathway activation by soluble IgG 150. Landerman, N. S. 1962. Hereditary aggregates. Immunology49:215-22 angioneurotic edema.J. Allergy 33: 137. Laurell, A. B., Martensson, 316-29 Sjoholm, A. G. 1979. Quantitation 151. Donaldson,V. H., Rosen, F. S. 1966. of Clr-Cls-CI inactivator complexes Hereditary angioneurotic edema: a by eleetroimmunoassay.Acta Pathol. clinical survey. Pediatrics37:1017-27 Microbiol. Scand.87C: 79-81 152. Frank, M. M., Gelfand, J. A., Atkin138. Hack, C. E., Hannema,A. J., Erenson, J. P. 1976.Hereditaryangioedema: berg-Belmer, A. J. M., Out, T. E., the clinical syndromeand its manAaberse,R. C. 1981. A Cl-inhibitoragement.Ann.intern. Med.84:580--93 complexassay (INCA): a method 153. Crowder,J. R., Crowder,T. R. 1917. detect C1 activation in vitro and in Five generations of angioneurotic vivo. J. lmmunol.127:1450-53 edema.Arch. Intern. Med.20:840-52 139. Nilsson, T., Back, O. 1985, Deter154. Austen, K. F., Sheffer, A. L. 1965. mination of Cls-C1 inhibitor comDetectionof hereditary angioneurotic plexes in plasmaby meansof an enzyme edemaby demonstration of a profound linked immunosorbentassay. Clin. reduction in the second componentof Exp. Immunol.60:178-82 humancomplement.N. Engl. J. Med. 140. Waldo,F. B., West, C. D. 1987. Quan272:649-56 titation of (CIINH]2Clr-Cls com- 155. Hadjiyannaki, K., Lachmann,P. J. plexesin glomerulonephritis as an indi1971. Hereditary angioedema:a review cation of C1activation. Clin. Immunol. with particular reference to pathoImmunopathol.42:239-49 genesisand treatment. Clin. Allergy I: 14l. Medicus,R. G., Chapuis, R. M. 198l. 221-33 The physiological mechanismof C1 156. Start, J. C., Brasher,G. W.1974.Eryactivation. Hoppe-Seyler’sZ. Physiol. thema marginatumpreceding hereditary angioedema.J. Allergy Clin. lmChem.362:30 (Abstr.) 142. Folkerd, E. J., Gardner, B., Hughesmunol. 53:352-55 Jones, N. C. 1980. The relationship 157. Blohme, G., Ysander, K., Korsanbetweenthe bindingability and therate Bengsten,K. 1972. Hereditary angloof activation of the complementcomneurotic oedemain three families. Acta ponent C1. Immunology41:179-85 Med, Scand. 191:209-19 143. Ziccardi, R. J. 1985.Demonstrationof 158. Ogston, D., Walker,J., Campbell,D. the interaction of native C1with monoM.1981. C1 inactivator level in pregmeric immunoglobulins and CI inhibinancy. Thromb.Res. 23:453-55 159. Donaldson,V. H. 1966. Seruminhibitor. J. Immunol.134:255943 144. Dodds,A. W., Sire, R. B., Porter, R. tor of C’Iesterasein healthanddisease. R., Kerr, M.A. 1978.Activationof the J. Lab. Clin. Med.68:369-82 first componentof humancomplement 160. Amir, J., Pensky, J., Ratnoff, O. D. (C1) by antibody-antigen aggregates. 1972. Plasmainhibition of activated plasma thromboplastin antecedent Biochem.J. 175:383-90 (factor XIa) in pregnancy.J. Lab. Clin. 145. Kasahara, Y., Takahashi, K., Nagasawn, S., Koyama,J. 1982. Formation Med. 79:106-12 of a conformationallychangedClr, a 161. Gordon,~E. M., Ratnoff, O. D., Saito, subcomponentof the first component H., Donaldson, V. H., Pensky, J., of humancomplement, as an interJones, P. K. 1980. Rapidfibrinolysis, augmented Hagemanfactor (factor mediateof its autoactivationreaction. FEBSLett. 141:128-31 XII) titers, and decreasedC1esterase inhibitor titers in womentaking oral 146. Niinobe, M., Yoshihiko,U., Hitonii,

Annual Reviews

Annu. Rev. Immunol. 1988.6:595-628. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

C1 INHIBITOR 625 contraceptives. J. Lab. Clin. Med.96: oneurotic edema(H.A.N.E.) plasma. 762-69 Trans. Assoc. Am.Physicians90: 174162. Donaldson,V. H., Evans, R. R. 1963. 83 A biochemical abnormality in here174. Strang, C. J., Spragg,J., Cholin,S., ditary angioneurotic edema.Am. ,I. Davis, A. E. III. 1987. Kinin-like Med. 35:37--44 activity of a peptide derived from complementcomponentC2. Fed. Proc. 163. Donaldson,V. H., Rosen,F. S. 1964. Action of complementin hereditary 46:1196(Abstr.) angioneurotic edema:the role of C’I 175. Curd,J. G., Prograis, L. J. Jr., Cochesterase. J. Clin. lnvest. 43:2204-13 rane, C. G. 1980. Detection of active 164. Carpenter,C. B., Ruddy,S., Shehadeh, kallikrein in inducedblister fluids of hereditary angioedema patients. J. Exp. I. H., Muller-Eberhard, H. J., Merrill, J. P., Austen,K. F. 1969. Complement Med. 152:742-47 metabolismin man: hypercatabolism 176. Schapira, M., Silver, L. D., Scott, C. F., Schmaier,A.H., Prograis,L. J. Jr., of the fourth (C4) and third (C3) ponentsin patients with renal allograft Curd, J. G., Colman,R. W.1983. Prerejection and hereditary angioedema kallikrein activation and high-molecular-weight kininogenconsumptionin (HAE).J. Clin. Invest. 48:1495-1505 hereditary angioedema. N. En#l. J. Med. 165. Laurell, A. B., Lindegren,J., Malmros, I., Martensson,H. L. 1969. Enzymatic 308:1050-53 and immunochemical estimation of C 1 177. Zuraw,B. L., Curd, C. G. 1986. Demesterase inhibitor in sera frompatients onstration of modifiedinactive first with hereditary angioedema.Scand. J. component of complement (C 1) inhibitor in the plasmas of CI inhibitorClin. Lab. Invest. 24:221-25 deficient patients. J. Clin. Invest. 78: 166. Donaldson, V.H. 1968. Mechanismsof activation of C1esterase in hereditary 567-75 angioneurotic edemaplasmain vitro. 178. Bjork,I., Jackson,C. M., Jarnvell, H., The role of Hagemanfactor, a clotLavine,K. K., Nordling,K., Salsgiver, promoting agent. J. Exp. Med. 127: W.J. 1982. The active site of antithrombin.Releaseof the sameproteo411-29 167. Ghebrehiwet, B., Silverberg, M., lytically cleavedformof the inhibitor Kaplan, A. P. 1981. Activation of the fromcomplexeswith factor IXa, factor classical pathway of complementby Xa, and thrombin.J. Biol. Chem.257: Hagemanfactor fragment. J. Exp. 2406-11 Med. 153:655-76 179. Nilsson, T. 1983. Onthe interaction 168. Ghebrehiwet, B., Randazzo, B. P., betweenhumanplasma kallikrein and Dunn,J. T., Silverberg,M., Kaplan,A. Cl-esterase inhibitor. Thromb.HaemoP. 1983. Mechanismof activation of stas. 49:193-95 the classical pathwayof complement 180. Johnson, D.A.,Travis, J. 1976.Human by Hageman factor fragment. J. Clin. alpha-l-proteinase inhibitor mechInvest. 71:1450-56 anismof action: evidencefor activation 169. Kaplan, A. P., Austen, K. F. 1970. A by limited proteolysis. Biochem.Biopre-albumlnactivator of prekallikrein. phys. Res. Commun.72:33-39 J. lmmunol.105:802-11 181. Aubry,M., Bieth, J. 1977. Kinetics of 170. Revak,S. D., Cochrane,C. G., Bouma, the inactivation of humanand bovine trypsins and chymotrypsinsby l-proB. N., Griffin, J. H. 1978.Surfaceand fluid-phase activities of two formsof teinase inhibitor and of their reacactivated Hagemanfactor produced tivation by 2-macroglobulin. Clin. duringcontact activation of plasma.J. Chim.Acta 78:371-80 Exp. Med. 147:719-29 182. Jornvall, H., Fisk, W.W., Bjork, I. 171. Goldsmith,G. H., Saito, H., Ramoff, 1979. The thrombin cleavage site in O. D. 1978. The activation of plasbovine antith~’ombin.FEBSLett. 106: minogen by Hagemanfactor (factor 358-62 XII) and Hageman factor fragments.J. 183. Laine, A., Davril, M., Hayem,A., LouClin. Invest. 62:54-60 cheux-Lefevbre, M.-H. 1982. Com172. Colman, R. W. 1969. Activation of parison of the interactions of human1plasminogenby humanplasma kalliantichymotrypsin with humanleukokrein. Biochem.Biophys.Res. Commun. cyte cathepsin G and bovine chymo35". 273-79 trypsin. Bioehem.Biophys. Res. Com173. Donaldson,V. H., Rosen,F. S., Bing, mun. 107:337-44 D. H. 1977. Role of the second com- 184. Donaldson, V. H., Harrison, R. A., ponent of complement(C2) and plasRosen,F. S., Bing, D. H., Kindness, minin kinin release in hereditaryangiG., Canar,J., Wagner,C. J., Awad,S.

Annual Reviews

Annu. Rev. Immunol. 1988.6:595-628. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

626

DAVIS

1985. Variability in purified dysfunctional Cl-inhibitor proteins from patients with hereditary angioneurotic edema:functional and analytical gel studies. J. Clin. Invest. 75:124-32 185. Alsenz, J., Bork, K., Loos, M. 1987. Autoantibodyomediated acquired deficiencyof C1inhibitor. N. Engl. J. Med. 316:1360-66 186. Curd, J. G., Yelvington,M., Burridge, N., Stimler,N.P., Gerard,C., Prograis, L. J., Cochrane,C. G., Muller-Eberhard, H. J. 1982.Generationof bradykinin during incubation of hereditary angioedemaplasma. Mol. lmmunol.19: 1365(Abstr.) 187. Fields, T., Ghebrehiwet, B., Kaplan,A. P. 1983. Kinin formationin hereditary angioedemaplasma: evidence against kinin derivative fromC2and in support of "spontaneous"formation of bradykinin. J. AllergyClin. Immunol.72: 54-

E., Bing,D. E. 1977.Inhibition of Clsinducedvascular leakagein guineapigs by substituted benzamidineand pyridinium compounds.J, Immunol. 118: 466-71 196. Davis, A. E. III, Davis, J. S. IV, Rabson,A. R., Osofsky,S. G., Colten, H. R., Rosen,F. S., Alper,C. A. 1977. Homozygous C3 deficiency: detection of C3 by radioimmunoassay. Clin. lmmunol,lmrnunopathol.8:543-50 197. Strang, C. J., Auerbach,H. S., Rosen, F. S. 1986. Cls-induced vascular permeability in C2-deficient guinea pigs. J. Immunol.137:631-35 198. Smith,M.A., Kerr, M.A. 1985.Cleavage of the second componentof complementby plasmaproteases: implications in hereditary Cl-inhibitor deficiency. Immunology56:561-70 199. Rosen,F. S., Alper, C. A., Pensky,J., Klemperer, M. R., Donaldson, V. H. 1971, Genetically determinedhetero188. Smith, M. A., Kerr, M. A. 1983. The geneity of the C1esterase inhibitor in kinin activity generated in CI 1NHpatients with hereditary angioneurotic deficient plasmais not derivedfromC2 edema.J. Clin. Invest. 50:214349 and originates from the kallikrein200. Gadek,J. E., Hosea,S. W.,G-elfand,J, kininogenpathway.Immunobiology 164: A., Frank, M. M. 1979. Response of 299 (Abstr.) variant hereditary angioedemapheno189. Donaldson,V. H., Ratnoff, O. D., Dias types to danazol therapy. Genetic da Siva, W., Rosen,F. S. 1969. Permeimplications.J. Clin. Invest. 64:28I)-$6 ability-increasingactivity in hereditary 201. Harpel,P. C., Hugli, T. E., Cooper,N. angioneurotic edemaplasma. J. Clin. R. 1975. Studies on humanplasma C1 Invest. 48:642-53 inactivator-enzymeinteractions. II. Structural features of an abnormalC1 190. Donaldson, V. H. 1973. Kinin forraation in hereditary angioneurotic inactivator froma kindred with hereedema (HANE)plasma. Int. Arch. ditary angioneurotic edema.J. Clin. Allergy 45:206-9 Invest. 55:605-11 191. Donaldson,V. H., Metier, E., Rosen, 202. Curd, J. G., Yelvington,M., Ziccardi, F. S., Kretschmer,K. W., Lepow,I. H. R. J., Mathison,D. A., Griffin, J. H. 1970.A polypeptidekinin in hereditary 1981.Purification and characterization angioneurotic edemaplasma: role of of twofunctionallydistinct formsof C1 complementin its formation. J. Lab. inhibitor from a patient with angioedema.Clin. Exp. Immunol.45:261-70 Clin. Med.76:986(Abstr.) 192. Davies,G. E., Lowe,J. S. 1960.A per- 203. Laurell, A. B., Martensson,U. 1971. meability factor released from guinea C1 inactivator protein complexedwith pig serum by antigen-antibody prealbuminin plasmafrom a patient with cipitates. Br. J. Exp. Pathol. 41: 335angioneurotic edema.Eur. J. Immunol. 1:14649 193. Klemperer, M. R., Donaldson,V. H., 204. Harrison, R. A., Rosen, F. S. 1982. Structural characterizationof Cl-esterRosen,F. S, 1968.Effect ofC’l esterase on vascular permeabilityin man:studase inhibitor (CI-INH) and comparison with dysfunctional proteins ies in normaland complement-deficient individuals and in patients with herefrom individuals with HANE.Mol. ditary angioneurotic edema.J. Clin. Immunol.19:1374(Abstr.) Invest. 47:604-11 205. Shokeir, M.H. K. 1973. Thegenetics of hereditary angioedema:a hypothesis. 194. Ratnoff, O. D., Lepow,I. H. 1963. Complement as a mediator of inflamClin. Genet.4:494-99 mation. Enhancement of vascular per- 206. Lachmann,P. J., Rosen, F. S. 1978. meabilityby purified humanC’I esterGenetic defects of complement in man. Springer Semin. lmmunopathol.1: 339ase. J. Exp. Med.118:681-98 53 195. Andrews,J. M., Rosen,F. S., Silverberg, S. J., Cory,M., Schneeberger,E. 207. Lachmann,P. J., Rosen, F. S. 1984.

Annual Reviews

Annu. Rev. Immunol. 1988.6:595-628. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

Cl

Thecatabolismof Cl-inhibitor andthe pathogenesis of hereditary angloedema.Acta Pathol. Microbiol. Immunol. Scand.(Suppl.) 284:35-39 208. Quastel,M., Harrison,R., Cicardi, M., Alper, C. A., Rosen, F. S. 1983. Behavior in vivo of normal and dysfunctional C1inhibitor in normalsubjects and patients with hereditary angioneurotic edema.J. Clin. Invest. 71:1041-46 209. Stoppa-Lyonnet, D., Tosi, M., Laurent, J., Sobel, A., Lagrue,G., Meo, T. 1987.Altered C1inhibitor genesin type I hereditary angioedema.N. Engl. J. Med. 317:1-6 210. Cicardi, M., Igarashi, T., Kim,M.S., Frangi, D., Agostoni,A., Davis, A. E. III. 1987. Restriction fragmentlength polymorphism of the C1 inhibitor gene in hereditary angioneurotic edema.J. Clin. Invest. In press 211. Cicardi, M., Igarashi, T., Kim,M.S., Frangi, D., Agostoni,A., Rosen,F. S., Davis,A. E. III. 1987.Geneticheterogeneity of hereditary angioneurotic edema.Clin. Res. 35: 606A(Abstr.) 212. Spaulding, W. B. 1960. Methyltestosteronetherapyfor hereditaryepisodic edema(hereditary angioneurotie edema).Ann. Intern. Med.53:739-45 213. Granerus,G., Hallbers, L., Laurell, A. B., Wetterquist,H. 1967.Studieson the histamine metabolismand the complementsystemin hereditary angioneurotic edema.Acta Med.Sca~fd.182:11-22 214. Willms,K., Rosen,F. S., Donaldson, V. H. 1975. Observationson the ultrastructure of lesions inducedin human andguinea pig skin by C1esterase and polypeptidefrom hereditary angioneuroti¢ edema (HANE)plasma. Clin. Immunol.Immunopathol.4:174-88 215. Davis,P. J., Davis,F. B., Charache,P. 1974. Long-termtherapy of hereditary angioedema (HAE).Preventive management with fluoxymesterone and oxymetholone in severely affected malesand females. JohnsHopkinsMed. J. 135:391-98 216. Rosse,W.F., Logue,G. L., Silverman, H. R., Frank, M. M. 1976. Theeffect of synthetic androgensin hereditary angioneurotic edema:alteration of C1 inhibitor andC4 levels. Trans. Assoc. Am. Physicians 89:122-32 217. Sheffer, A. L., Fearon,D. T., Austen, K. F. 1977. Methyltestosteronetherapy in hereditary Ann.Intern. Med. 86:306-8angioedema. 218. Gelfand,J. A., Sherins, R. J., Ailing, D. W., Frank, M. M. 1976. Treatment of hereditary angioedema with danazol.

INHIBITOR

627

Reversal of clinical and biochemical abnormalities. N. Engl. J. Med. 295: 1444-48 219. Hauptmann,G. 1978. Le traitemente des deficits de l’inhibiteur de la C1esterase par le Danazol.Med.Hyg.36: 2569-75 220. Pitts, J. S., Donaldson, V. H., Forristal, J., Wyatt, R. J. 1978. Remissions induced in hereditary angioneurotic edema with an attenuated androgen (danazol): correlation betweenconcentration of Cl-inhibitor and the fourth and second components of complement.J. Lab. Clin. Med. 92: 501-7 221. Sheffer, A. L., Fearon,D. T., Austen, K. F. 1979. Clinical and biochemical effects of impeded androgen (oxymetholone) therapy of hereditary angioedema.J. Allergy Clin. Immunol. 64:275-80 222. Agostoni,A., Cicardi, M., Martignoni, C.,Bergamaschini, L., Marasini, B. 1980. Danazoland stanazol in longterm prophylactic treatment of hereditary angioedema.J. Allergy Clin. Imrnunol.65:75-79 223. Sheffer, A. L., Fearon, D. T., Austen, K. F. 1981. Clinical and biochemical effects of stanazolol therapyfor hereditary angioedema.J. Allergy Clin. Immunol. 68:181-87 224. Costanzi,J. J., Coltman, J. J. Jr., Donaldson, V. H. 1969. Activation of complement by a monoclonal cryoglobulin associated cold urticaria. J. Lab. Clin. Med.with 74:902-10 225. Caldwell,J. R., Ruddy,S., Schur, P. H., Austen, K. F. 1972. AcquiredC1 inhibitor deficiency in lymphosarcoma. Clin. Immunol.lmmunopathol.1:39-52 226. Day, N. K., Winfield, J. B., Gee, T., Winchester,R., Teshima,H., Kunkel, H. G. 1976. Evidencefor immune complexes involvinganti-lymphocyteantibodies associated with hypocomplementemia in chronic lymphocytic leukaemia(CLL). Clin. Exp. Immunol. 26:189-95 227. Hauptmann,G., Lang, J. M., North, N. L., Obeding,F., Mayer,G., Lachmann,P. J. 1976.AcquiredC1inhibitor deficienciesin lymphoproliferative diseases with serum immunoglobulinabnormalities. Blur 32:195-206 228. Schreiber,A. D., Zweiman, B., Atkins, P., Goldwein, F., Pietra, G., Atkinson, B., Abdou,N. I. 1976. Acquiredangioedemawith lymphoproliferative disorder: association of C1 inhibitor deficiency with cellular abnormality. Blood 48:567-80

Annual Reviews

Annu. Rev. Immunol. 1988.6:595-628. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

628

oAvis

229. Oberling, F., Hauptmann,G., Land, 13. M., Bergerat,J. P., Mayer,G., Batzenschlager, A., Hamman, B., Gillett, B. 1975.Deficits acquisde l’inhibiteur de la C1 esterase au cours de syndrome lymphoidcs. Nouv. Presse Med. 4: 2705-8 230. Cohen,S. H., Koethe, S. M., Kozin, F., Rodey,G., Arkins,3. A., Fink,3. N. 1978. Acquiredangioedemaassociated rectal carcinomaand its response to danazoltherapy. J. Allergy Clin. Immunol. 62:217-21 231. 13elfand,J. A., Boss,G. R., Conley,C. L., Reinhart, R., Frank, M.M. 1979. Acquired CI esterase inhibitor deficiency and angioedema:a review. Medicine 58:321-28 232. Kondo,M., Yokoe,N., Nishibori, H., Takemura,S., Yoshikawa,T., Kato, H., Hosokawa,K., Abe, T. 1978. A case of secondary CI inhibitor deficiency associated with benign monoclonal gammopathyand angioneurotic edema.RinshoKetsuki 19: 158187 233. Hauptmann,G., Petitjean, F., Lang, J. M., Oberling, F. 1979. AcquiredC1 inhibitor deficiencyin a case oflymphosarcoma of the spleen. Reversal of complement abnormalitiesafter splenectomy.Clin. Exp. Immunol.37: 52331 234. Fiechtner,J. J., Marx,J. J. Jr., Walski, K. P., Schloessner, L. L. 1980. Acquired angioedema, autoimmune hemolyticanemia, and lymphoma: resolution after therapy. Clin. Immunol. Immunopathol.15:642-45 235. Cicardi, M., Frangi, D., Bergamaschini, L., Gardinale, M., Sacchi, G., Agostoni, A. 1985. Acquired C1 inhibitor deficiency with angioedema

symptomsin a patient infected with Echinococcusgranulosus. Complement 2:133-39 236. Sheffer, A. L., Austen,K. F., Rosen, F. S., Fearon, D. T. 1985. Acquired deficiencyof the inhibitor of the first componentof complement:report of five additional cases with commentary on the syndrome. J. Allergy Clin. Immunol. 75:640-46 237. Geha,R. S., Quinti, I., Austen,K. F., Cicardi, M., Sheffer, A., Rosen,F. S. 1985. AcquiredCl-inhibitor deficiency associated with antiidiotypic antibody to monoclonal immunoglobulins. N. Engl. J. Med. 312:534-40 238. Fust, G., Czink,E., Minh,D., Mizlay, Z., Varga, L., Hollan, S. R. 1985. Depressedclassical complementpathwayactivities in chronic lymphocytic leukaemia. Clin. Exp. Immunol. 60: 489-95 239. Jackson, J., Sire, R. B., Whelan,A., Feighery, C. 1986. AnIgG autoantibody whichinactivates Cl-inhibitor. Nature 323:722-24 240. Melamed, J., Alper, C. A., Cicardi, M., Rosen,F. S. 1986. Themetabolismof C1inhibitor and Clq in patients with acquired Cl-inhibitor deficiency. J. Allergy Clin. Immunol.77:322-26 241. Frank, M. M., Malbran, A., Simms, H., Melez,K., Santaella, M., Hammer, C., Fries, L. 1987. Acquiredangioedematype II: a newautoimmune disease. Clin. Res. 35: 641A(Abstr.) 242. Malbran, A., Hammer,C., Frank, M. M., Fries, L. 1987. Acquired angioedematype II: mechanism of action of an autoantibody directed against C1 esterase inhibitor. Clin. Res. 35: 255A (Abstr.)

Annu. Rev. Immunol. 1988.6:595-628. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

Annu. Rev. Immunol. 1988.6:595-628. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

Annual Reviews www.annualreviews.org/aronline

Annu. Rev. Immunol. 1988.6:629-662. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

Ann. Rev. Immunol. 1988.6:62942 Copyright©1988by AnnualReviewsInc. All rights reserved

THE T CELL RECEPTOR/CD3 COMPLEX: A DYNAMIC PROTEIN ENSEMBLE Hans Clevers, Balbino Alarcon, Thomas Wileman, and Cox Terhorst Laboratory of Molecular Immunology, Dana-Farber Cancer institute, 44 Binney Street, Boston, Massachusetts 02115

T CELL RECEPTORS DO NOT ACT ALONE A large body of information about antigen receptors on the surface of T lymphocyteshas been gathered in the last five years (1, 2). T cell receptors use a variable region gene pool that is completelydistinct from the variable genes of immunoglobulins. Indeed, T cells recognize different antigenic entities than do B lymphocytes. The latter notion was most dramatically demonstrated by the manyobservations that led to the conclusion that T cell receptors corecognize processed nominal antigen and a 9ene product of the MHC(3). Since T cell receptors (TCR) and MHCproducts anchored in the plasma membrane of T lymphocytes and antigen-presenting cells, respectively, the TCR/antigen/MHC recognition takes place on the interface between the two cells. A localized and TCR-independent adhesion provides a stabilizing environment for the subtle ternary interaction, which is dependent upon the fine recognition of all three of its participants. Frommodel studies with humancytotoxic T cells, it appears that this transient adhesion event is initiated prior to the interaction of TCRwith antigen and MHC(4). The T cell receptors for antigen and MHCconsist of two disulfidelinked variable glycoproteins whosegenes rearrange in T cells: the T cell receptor (TCR)~ and // chains (1, 2). The T cell receptor so defined subserves both antigen and MHC recognition. Cell fusion experiments (5) and transfection of TCR-eand -/~ chain cDNAsof defined specificities between T cell clones (6) confirm that the e//~ heterodimer confers both 629 0732~582/88/041043629502.00

Annual Reviews

Annu. Rev. Immunol. 1988.6:629-662. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

630

CLEVERS ET AL

antigen and self-MHCspecificity upon the cell that expresses it. Recently, humanand murine T cell clones have been described that express other receptor molecules, TCR-v/3(1, 2, 7, 8) or TCR-v/V(9) on their surfaces. Althoughsomeof those cells have natural killer (NK)-like activities, the function of TCR-~+/6+ lymphocytes has not been elucidated and their target antigens (TCR-v/6 ligands) are unknown.None of the TCR-v/6 TCR-v/Vkiller T cell clones acts in an MHC-restrictedfashion; the current hypothesis therefore is that only TCR-~/fl receptors corecognize MHC and antigen. The molecular basis of the recognition of MHCand MHC/antigen is not yet established. A large body of experimental evidence nowfavors the notion that the final T cell repertoire is selected from a pool of randomly generated TCRsbased on MHCrecognition (10, 11). Hedrick and colleagues (12) recently examinedTCR-~and -fl gene expression in a series of class II MHC alloreactive T cell clones and in antigen-specific clones restricted by the same class II MHC elements. They found that the same V~ and Vfl gene segments were used. Apparently, TCRsmediating allorecognition of Ia antigens are homologousor identical to TCRsinvolved in recognition of the sameIa antigens in association with nominalantigen. This suggests that MHC-restricted recognition and alloreaction are not distinct events but rather represent differences in the affinity of TCR/MHC interactions. Since recognition of nominal antigen and MHCby TCRmost likely takes place after an adhesion occurs between a T cell and an antigen presenting cell, several T cell surface glycoproteins formerly called "accessory molecules" have turned out to be adhesion structures. Primary adhesion molecules on the surface of T lymphocytes are the cell surface proteins CD2and LFA-1(13). Convincing experimental evidence supports the idea that CD2molecules on the T cell bind with high affinity to LFA3 embeddedin the plasma membraneof the juxtaposed antigen-presenting cell. A molecule termed I-CAMis probably one of the cell-bound ligands for the LFA-1structure. CD8and CD4most likely interact with a nonpolymorphicregion of the class I and class II MHC molecules, respectively (1, 14). Not only are the postulated interactions between CD4/CD8and MHCstructures worthy of in-depth molecular studies, so is the implied proximity of CD4or CD8and TCRs. The latter type of transient interactions maybe of crucial importancefor the triggering of T cell.functions (15, 15a). Interactions of the adhesion molecules with their respective ligands not only stabilize cell/cell contact, they also send either positive or negative signals to the T cell proliferative pathway induced by TCR/antigen/MHC interactions. The appropriate antibodies directed at CD2can induce T cell

Annual Reviews THE TCR/CD3 COMPLEX

631

Annu. Rev. Immunol. 1988.6:629-662. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

INSIDI~

P CD3-~

P

P

P ~

7

~-

P~I

Figure 1 The TCR/CD3complex indicates N-linked glycosylation. S-S indicates sulfhydryl bridges. P indicates sites of phosphorylation.

proliferation even in cells that do not express the TCRon their cell surface. Bycontrast, antibodies directed at CD4and CD8only cause T cell activation in combinationwith anti-TCR/CD3 reagent (15). TCRsdo not appear to act alone in the complexpathwaysof antigendriven T cell proliferation. Instead, they operate within a networkof interactions that is only partially understood.TCRsare thoughtto transmit the signal providedby ligand/receptor interaction across the plasma membrane.To this end, the variable, or clonotypic, TCRall3 or 7/6 heterodimers mayrelay that signal through the invariant CD3proteins (CD3-7, 6, 8, and ~). Together, these polypeptide chains form the TCR/CD3 complex(Figure 1). Since this TCR/CD3 complextransiently associates with other proteins like CD3-p21and perhaps with CD4or CD8,the TCR/CD3 complexoperates as a dynamicensemble of proteins whosemembersdiffer dependingon its functional state. Our current knowledgeof its structure, assembly,and function are discussed in this review. CURRENT VIEWS ON THE STRUCTURE T CELL RECEPTOR/CD3 COMPLEX

OF THE

The TCR/CD3 Complex on the Cell Surface Antigen receptors on the surface of humanand mouseT lymphocytes have been discovered through two lines of experimentalresearch. First, monoclonalantibodies (MAbs) specific for functionally competentin vitro cultured T cell clonesweremade(16, 17). Theseclone-specificor clonotypic antibodies were shownto affect the function of their respective T cell

Annual Reviews

Annu. Rev. Immunol. 1988.6:629-662. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

632

CLEVERS ET AL

clone and were used to characterize these antigen receptors biochemically. Second, genes were sought that specifically rearrange only in T ceils. The T-cell-specific, rearranging, variable gene families give rise to a large number(> 10~1) of different T cell receptors for antigen (1, 2, 18, 19) and the gene families code for those structures recognized by the clonotypic antibodies. Although clonotypic antibodies react with TCRheterodimers (ct/fl or 7/3) or homodimers(~/~), under mild extraction conditions they can be used for isolation of the TCR/CD3complex from the plasma membrane. The same complement of CD3proteins is associated with TCR-a/fl and TCR-7/6 heterodimers (9, 20-23). The CD3antigen was first identified as a 20-kd glycoprotein, present on the surface of all humanT lymphocytes, by immunoprecipitation with the monoclonal antibody OKT3(24). As different labeling and immunoprecipitation methods were used, the structure of the CD3antigen appeared more complex. Thus, soon after, the CD3antigen was found to be expressed as a complex composedof the major glycoprotein of 20 kd, a 25-28 kd glycoprotein, and two minor glycoproteins of 37 and 44 kd (25, 26). The existence of a 20 kd nonglycosylated protein was suspected, since upon treatment with endoglycosaminidases H and F (Endo H and F), a portion of the 20-kd proteins remainedundigested (26-28). Definitive proof for a second 20-kd protein was obtained when trypfic pepfide maps and partial N-terminal sequences of both proteins were compared(29) and when monoclonal antibodies were raised that distinguished between the two 20-kd CD3proteins by immunoblotting (30). At this point the largest CD3proteins, the 44-kd and 37-kd polypeptides, were identified as the TCR-aand -fl chains (31, 32). The 25-28 kd glycoprotein was therefore termed CD3-7; the 20-kd glycoprotein, CD3-6; and the 20-kd nonglycosylated protein CD3-e(28, 29). Several lines of evidence support the concept that the TCR-a/fl heterodimer is associated with the CD3proteins (Figure 1). First, coprecipitation of the CD3pr6teins with anti-TCR reagents and vice versa indicated a close proximity of these cell surface structures. Second, incubation of T cells with MAbsdirected against either TCRor CD3, or incubation of antigen-specific cloned T cells with antigenic peptides, leads to "co-modulation" or simultaneous disappearance from the cell surface of both structures (33, 34). Third, several mutants that do not express the TCR/CD3 complex on their surface have been analyzed and found to lack the TCRa or TCR-fl mRNA’s.Upon gene transfer of either TCR-a or TCR-fl, cDNA’s cell surface expression of the TCR/CD3complex could be restored to normal levels (35, 36). More recent studies of the murine TCR/CD3complex added new members to the CD3family (Figure 1 and Table 1). Murine TCR-associated

Annu. Rev. Immunol. 1988.6:629-662. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

Annual Reviews THE TCR/CD3 COMPLEX 633

Annual Reviews

Annu. Rev. Immunol. 1988.6:629-662. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

634

CLEVERS ET AL

CD3proteins were isolated with anti-TCR MAbs(37, 38). Thus, a complex of five CD3-1ikeproteins has been identified, consisting of two N-glycosylated proteins of 21 and 28 kd, one glycoprotein which maycontain Olinked oligosaccharides (25 kd) (38a), one nonglycosylated phosphoprotein (21 kd), and a nonglycosylated disulfide-bridged homodimer(17 kd) 38). The 28-kd glycoprotein reacts with an antibody raised against peptide deduced from the cDNAsequence of murine CD3-~i (38) and therefore the murine equivalent of CD3-~. The 25-kd non-N-glycosylated protein was assigned as the murine counterpart of humanCD3-e by reaction with sPr, a monoclonal antibody raised against human CD3-~(30; B. Alarcon, C. Terhorst, unpublished) and by transfection experiments (38a). The 21-kd glycoprotein was assigned as the murine equivalent CD3-~, by comparison with the molecular weight and the single N-linked oligosaccharide predicted from the amino acid sequence. The 34-kd nonglycosylated homodimeris a protein not previously found in humancells and is called CD3-((37, 38). Anantibody raised against this murine protein detected a humanCD3-(, which comodulated with the TCR~/fl chains. However, humanCD3-( could not be isolated by co-immunoprecipitation with anti-TCR or anti-CD3 reagents (39). An additional 21-kd nonglycosylated murine protein (CD3-p21), phosphorylated at a tyrosine residue, is sometimes disulfide-linked to CD3-( (40, 41). This contrasts with the 21-kd glycoprotein (CD3-7) and 25-kd CD3-e which are phosphorylated at serine residues upon treatment with PMAand afitigen (38). Thus, studies of the murine TCR/CD3complex have contributed substantially to our knowledge of this multicomponent membraneprotein system. It is possible that other membraneproteins weakly associated with the known TCR/CD3complex may be discovered. Moreimportantly, it remains to be determined which cytoplasmic proteins are--albeit transiently--associated with the TCR/CD3 complex. Structure

of the Individual

Polypeptide

chains

a-CR-~/# The molecular weights of the humanTCR-~and -fl chains vary in size (~ from 43 kd to 49 kd; fl from 38 to 44 kd) (1, 2) (Table 1). TCR-~and -fl are heterogeneously charged glycoproteins; TCR-~contains N-linked oligosaccharides of the complex type, whereas TCR-fl contains both high mannose and complex N-linked glycan side chains (I, 27, 28). The polypeptide backbones for TCR-~and -fl are 32 kd to 34 kd, respectively. The murine TCR-~/fl is also a heterodimer composedof 40- to 50kd TCR-~and -fl glycoproteins which do not resolve well by SDS-PAGE. The murine TCR-~and TCR-fl chains contain N-linked oligosaccharides attached to polypeptide backbones of 28 and 32 kd respectively (1). As humans, the murine TCR-~ chain contains only complex type glycans,

Annual Reviews

Annu. Rev. Immunol. 1988.6:629-662. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

THE TCR/CD3

COMPLEX

635

whereas the TCR-/~chains contain two or three high mannoseglycans (1, 2). Theprimaryaminoacid structure of the TCR-aand TCR-/~(2) proteins showsan immunoglobulin-like organization (Figure 2). Avariable region (V) and a constant region (C) are linked by a short joining segment in some of the genes, D segments are used. The TCRproteins have a transmembraneregion and a short cytoplasmic tail (5-12 aminoacids). Twocysteine residues in the V and C regions indicate an intrachain disulfide loop in each region. TCR-aand TCR-/3have cysteine residues, proximalto the transmembrane region, that form an interchain disulfide bond. UsingX-ray crystallographic data of immunoglobulins, predictions for the secondary structure of the V and C domains of TCR-aand /~ suggestthat they havea basic Ig fold structure (42). Secondarystructure predictions also suggest that the transmembrane domainof the TCR-aand -/~ chains maybe in the a-helical configuration. Assumingan amphipathic a-helix, two positive charges can be accommodatedin the putative 20 aminoacid transmembrane segment. Since the TCR-//transmembrane domainhas a lysine and the CD3-?,-6, and -5 each contains either aspartic acid or glutamicacid in that domain,interactions betweenthese membrane proteins maybe stabilized by salt bridges between the chargedaminoacid residues. TCR-?/6Althoughthe TCR-ygene was found to be rearranged and tran-

VARIABLE

REGION

]E

CONSTANT

Clio C-N ~ C--N-’--

TCRa, H

[~c,

L

N-C

Clio

ClioCliO

N’C-N--

CliO

L TCR).

CHO

REGION

C ~ N[~}--,,

CHO C--N--C

CliO

CHOCliO TM --~-

CHO

CC’~

TM

Figure 2 Schematic outlines of TCR-~t,fl, ?, and -6 polypeptide chains derived from Kabat (90) and Chien et al (50). Boxedareas represent hydrophobic regions. L, leader sequence, TM, transmembrane region.

Annual Reviews

Annu. Rev. Immunol. 1988.6:629-662. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

636

CLEVERS ET AL

scribed in mouse(43, 44) and human(45) T lymphocytes,the nature the TCR-yprotein product has been a mystery for sometime. A number ÷ T cell clones with NKactivity lack reacof humanCD4-, CD8-,CD3 tivity with WT31,a monoclonal antibody that recognizes a common epitope on humanTCR-~//~chains (7, 8, 9, 46). A heterodimercomposed of a 55-kd and a 40-kd protein was coprecipitated with anti-CD3 antibodies. Since the cells expressed neither TCR-~nor TCR-/3mRNA but did express TCR-7mRNA,the new CD3-associated proteins were candidates for a TCR-ygene product. By immunoprecipitation with an antipeptide anti-TCR-7antibody, the 55-kd protein was demonstratedto be the TCR-7product, while the 40-kd protein was a new TCR-like ÷, WT31-, CD4-, CDS-, TCRpolypeptide termed TCR-6(46). The CD3 y/g+ lymphocyteswere demonstratedto constitute a small subpopulation of normal humanperipheral lymphocytes (0.5%o-10%)and thymocytes (0.2%-0.9%)(20). In contrast with the TCR-~//3polypeptides, the TCR-~,proteins are apparentl~ expressed in several forms. The 55-kd TCR-yglycoprotein has a 29-kd polypeptide backboneand is the product of the Cy2gene that lacks cysteine residues outside the transmembrane domain(47). Another humanconstant region gene, C~1, encodesa protein that can formdisulfide bridges. Theproducts of this geneindeed form covalently linked TCR-~/6 heterodimers. In addition to two types of TCR-~/6heterodimers, a ~,/~ disulfide-linked homodimer has beenfound in humanT cell clones (8, 9, 48). The TCR-y-6or TCR-y/ystructures are functional, since anti-CD3 antibodies stimulate the production of IL-2 in immaturehumanthymocytes, and inhibit NKactivity mediated by the TCR-y/6CD3complex (9, 21). Highly purified NKcells isolated from unmanipulatedmice and propagatedin culture with recombinantinterleukin-2 for short periods of time did not express mRNA coding for TCR-a,fl, 3’ or CD3-~,3, e. (48a). TCR-y/6polypeptide chains have also been found in murine CD4-, CD8-thymocytes(22, 23, 49) (Table 1). The TCR-yprotein is a 35-kd glycoprotein with a polypeptide backboneof 32 kd, whichis disulfidelinked to a 45-kd TCR-6.The polypeptide backboneof the TCR-6glycoprotein is 31 kd (49, 50). TheTCR-y and -3 polypeptidechains are similar to the TCR-~//3proteins (50). AgainV and C regions are found to have weakbut significant homologiesto Ig domains(Figure 2). TheC~and sequences have a lower degree (9-18%) of homologyto the members the Ig superfamilythan do the Cr and Ca constant regions (,-~40%). Like that of C,, the Ca transmembraneregion probably forms an amphipathic helix with one or two positively chargedresidues. Anestimate of the size of the TCR-7/6repertoire will have to await further sequenceanalyses.

Annual Reviews THE TCR/CD3 COMPLEX

637

More importantly, the class of antigens recognized by TCR-?/6 or TCR?/y needs to be determined.

Annu. Rev. Immunol. 1988.6:629-662. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

The CD3 Polypeptide

Chains

The primary structures of the humanand murine CD3-~,-6, and -e chains have been derived from nucleotide sequences of the respective cDNAs(5156). Anoutline of the structure of these protein moleculesis given in Figure 3. Each contains an N-terminal extracellular domain, a transmembrane segment, and a cytoplasmic domain. In the case of the CD3-7 and -6 glycoproteins, the presence of a consensus sequence for N-linked glycosylation in the amino-terminal segment indicates that this segment is located extracellularly (Figure 3). The vectorial organization of CD3-e the plasma membrane is derived from experiments with antipeptide reagents directed at the N-terminus of the mature protein. In the case of both the humanand the murine CD3-e, these anti-N-terminal peptide sera stain T lymphocytes in an indirect immunofluorescenceassay. In the case of the humanCD3-e, the antibody is mitogenic for peripheral blood T lymphocytes. (H. Clevers, C. Terhorst, unpublished.) The transmembrane region of all knownCD3proteins contains a negatively charged amino acid which may be one of the components that

L

CD3-(S M

TM

CliO CliO CliO L E~]r-----c N--N-C N--

CD3-(~H -2t

L CDS-Y H

L

Clio

[~-’~

C I~-’-~CI~ !

16

C~O

TM C-C.~

TM

-- C-C--I"--~ 52

72 75 ~0

Clio Clio -N--C,-N--

’ 8~8590 ’’ ! 6.5

I0~ *tO6

TM .6:

t60

Figure 3 Schematic outline of the protein sequences of humanand mouse CD3proteins. (Legends, see Figure 2.)

Annual Reviews

Annu. Rev. Immunol. 1988.6:629-662. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

638

CLEVERS ET AL

stabilize intersubunit interactions in the TCR/CD3 Complex(Figure 4). The proposed transmembranesegmentsof most of the CD-3proteins have a predicted e-helix configurationand form therefore amphipathichelixes. In humanand murine CD3-fi and CD3-e chains, the transmembrane domaincontains half cystine residues. Althoughfatty acid residues have often been found in covalent linkage to Cys residues in transmembrane segmentsof cell surface proteins, this posttranslational modificationhas never been observed in the case of the CD3-6or CD3-eproteins, but the possibility that a fatty acid chain is attached to the murineCD3-~ polypeptide cannot be excluded. Interestingly, a cDNA whichdoes not contain a transmembrane region caused by an alternative splicing event has been isolated from a variant of the JURKAT cell line (56a). Whether this form of CD3-6is secreted or whetherit assemblesinto the TCR/CD3 complexremains to be determined. Since the cytoplasmic regions of the CD3polypeptide chains are considerably longer than those of the TCRe, -/~, -y, and-6 chains, these portionsof the moleculespresumably play an important role in the interaction with cytoplasmiccomponentsthat are directly involved in the transduction of the antigen-bindingsignal: The available aminoacid sequencesdo not reveal any homologieswith phosphokinases or other enzymes knownto be involved in transmembrane signal amplification pathways.Since the CD3-ecytoplasmictail is completely different from its CD3-7and CD3-6counterparts (Figure 4), CD3e mayhavea distinct function in the signal transducingprocess. Thearea immediatelyC-terminal to the transmembraneregion in CD3-econtains an exceptionally high numberof basic aminoacids followed by a short stretch of prolines (Figure 4). This sequencearrangementwas also found in the muchlonger cytoplasmic tail of CD2and could suggest that CD2 and CD3-emayinteract with the sameproteins on the cytoplasmicside of the plasma membrane.Although the cytoplasmic tails of CD3-7 and -6 are highly homologous (Figure 4), a unique functional role of the cytoplasmic tail of CD3-yis indicated by the phosphorylationof one or two of its serine residues(see below). Muchless is knownabout the structure of the CD3-~and -p21 proteins (Table 1). CD3-~occurs as a nonglycosylated homodimerof 17 kd. several instances, a heterodimerconsisting of this 17-kdprotein and a 15kd nonglycosylatedpolypeptide chain has been detected (38). The 15-kd polypeptidemightbe a proteolytic fragmentof the 17-kd CD3-~that exists in vivo or that arose duringthe isolation procedures.Part of the CD3-ff proteins (~ 10%)were found to be disulfide-bridged to CD3-p21.The possibility that this disulfide-bridgeformationis inducedby activation of the TCR/CD3 complexand links the receptor to the proliferative pathway needs to be investigated. Isolation of cDNA clones encodingCD3-~and

Annual Reviews THE TCR/CD3

T3 -~M T3-~H T3 T3 T3-~H

D ...... DGNEEMGG QTN--KAK

D

COMPLEX

639

S S R

Q - " S I K ~I~! ! ! ! ! i~~ ...... ........ ,

Annu. Rev. Immunol. 1988.6:629-662. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

T3 T3-~H T3 T3-~M T3

T3 T3

T3-5~ T3-~. TRANSMEMBRANE REGION T3 T3-£H

EIN CIMIE~M~D V MSV

A T I V I VIDII C IiTIG GIL[L~-LL~V Y[Y ~ S

j ~Q~N C[I~E~LI~N AA T II I SIGllF~F A E I~S I F V~LJA~G~V YJF IIA T3-~

T3 -~M T3 -~H T3

125 K N E K A K A K P V T R G T G A G S E P E G Q N K E~P P P Y P N KNRKAKAKPVTRGAGAGGRQEG~NKE~ P PV P N %~ D c vr&~Qrsl~rXlSrD~KrQ~Trc~rs~rQ-C~-~IK~A-~ d ~r~

T~-8~ T3-SH

T3-~.M T3-~H T3 T3-~M T3-~H Figure 4 Comparison of the a~no acid sequences of the human and routine CD3-7, J and e chains (51-56). Dashed boxes indicate homologies between CD3-T and -6. Solid boxes indicate amino acids that are found in the same position in either CD3-e and in CD3-7 or CD3-6 or in all six polypeptide chains. Alignments at the N-termini are in part based upon the known intron/exon boundaries of the respective genes (87-89) as indicated by arrows. The negatively charged residues in the CD3 transmembrane regions are marked by a black triangle.

Annual Reviews

640

CLEVERS ET AL

CD3-p21is of crucial importance to further our knowledgeabout the structure/function relation of these molecules.

Annu. Rev. Immunol. 1988.6:629-662. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

BIOSYNTHESIS AND ASSEMBLY OF THE TCR/CD3 COMPLEX A T cell cannot recognize an antigen unless the TCR/CD3 complexis correctly assembledand transported to the plasmamembrane.Early evidence that the processes of assemblyand transport are complexcomes from the demonstration that T cell mutants lacking one of the TCRor CD3chains fail to transport the remainingchains to the cell surface (57, 58, 59; B. Alarcon,unpublished).Theexperimentsindicate that the T cell has a mechanism to prevent incompletereceptors fromarriving at the cell surface. Ananalogoussituation arises during intrathymicdifferentiation. The genes encoding the knownmembersof the TCR/CD3complex are expressedsequentially during the final stages of T cell maturation. CD3y, CD3-6,and CD3-eare synthesized by the earliest recognizable thymocytes, but the proteins remain inside the cell (see below). Uponfurther maturation, T cells begin to express TCR-/~chains intracellularly. The TCR/CD3 complex is only transported to the plasma membraneafter +, CD4 ÷, CD8 ÷ thymocytes synthesis of the TCR-echain. Whilst CD1 express low levels of the TCR/CD3 complexon their cell surface, "single + or CD8÷ express high levels of the receptor complex. positive" cells CD4 Theenvironmentof the thymusgland maytherefore influence the assembly process. Takentogether these observationssuggest that T cells exploit an intracellular transport pathwaythat prevents surface expressionof incomplete receptors during biosynthesis. The timing of the appearance of the TCR/CD3 complexat the cell surface during T cell maturation can thus be controlled by the expression of a single chain of the complex. To understandthis pathwaywehave started to study assemblyand transport of the receptorin detail. Posttranslational Modification of CD3Proteins The protein backbones of the humanCD3-yand -6 chains have been calculated from their predicted aminoacid sequencesto be 16 kd. Both chains receive two N-linked high-mannoseoligosaccharides (Table 1). The glycosylated humanCD3-6chain migrates as a 20-kd molecule. In contrast, the humanCD3-ychain appears as a 23-kd band early during biosynthesis, whichin chase-labeling experimentsmaturesto a protein of 25-28 kd (38a, 58). Removalof the two N-linked glycans from human CD3-6yields a protein backboneof 14 kd. This is at variance with its

Annual Reviews

Annu. Rev. Immunol. 1988.6:629-662. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

THE TCR/CD3

COMPLEX 641

predicted molecular weight of 16 kd. It was proposed that this discrepancy was caused by proteolytic processing of the C-terminus. This hypothesis has been refuted recently since antibodies directed at the extreme Cterminus of the predicted amino acid sequence recognized the 14-kd CD36 chain (B. Alarcon, unpublished). The nature of the processing event that reduces the apparent molecular weight of CD3-6 to 14 kd remains unknown. The human CD3-5 chain does not appear to undergo any posttranslational modification. The protein backbones of murine CD3-~, -6, and -5 are 16 kd, 16 kd, and 17 kd, respectively. Again, the CD3-~and -6 chains are glycoproteins and addition of one or three N-linked oligosaccharide raises their apparent molecular weights to 20 kd and 25 kd, respectively. These molecular weights have been confirmed by transfecting COS-cells with the cDNAsencoding the humanand murine proteins (38a). In T cells and in transfected COScells, the murine CD3-emigrates as a 25 kd protein. However,the predicted molecular weight, confirmed by in vitro translation experiments, is only 18 kd (54). Therefore, we conclude that routine CD3e undergoesan as-yet-unidentified posttranslational modification. The CD3-~, -3, and -e Chains Form a CD3 "Subcomplex" (Figure 5) Several experiments showthat a CD3-~,-6, -5 core can form in the absence of TCR-~and/or TCR-fl chains. The earliest complexof proteins that can be observed during pulse-chase labeling experiments is one composedof CD3-~, -6, and -5. The independent assembly of the CD3chains has also been demonstrated in T cell lines that lack TCR-~or TCR-fl chains (58), and in COScells transfected with the relevant cDNAs.It is interesting that CD3chains are not transported to the plasma membraneof COScells. By virtue of the high sequence homologies between human and mouse CD3 proteins, heterologous associations between these chains in COScells could be demonstrated (38a). Similarly, heterologous CD3complexes can form following the transfection of humanCD3-5into a murine T-T hybridoma (H. Clevers, unpublished). It is likely that the CD3proteins form a core to which the TCRchains bind. It now seems clear from studies using T cell mutants and variants, that TCR-~and TCR-fl are independently associated with CD3. In human TCR-~- lines and TCR-~- mutants, a subcomplex of CD3-~6ewith TCRfl can be detected. Similarly, CD3-~65-TCR-~ was found in a TCR-flmutant (58). The binding of TCR-~ and TCR-fl to CD3 might be prerequisite for disulfide-bridged heterodimer formation. The T cell clone HY-827-P19 does not synthesize CD3mRNAsbut does produce considerable amounts of TCR-~and -fl chains. No TCRheterodimers are

Annual Reviews

642

CLEVERS ET AL CD~

CD~

CD~

CD~tM)

Annu. Rev. Immunol. 1988.6:629-662. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

CD~)

GOLGI M~turatioa

[CD3(’~F--TCR

CELL SURFACE

Figure 5

The assembly pathway of the TCR/CD3complex (see text).

formed, even though they are found in the parent cell line HY-827 (B. Alarcon, unpublished). Based on the types of analyses described here, wefavor a modelof hierarchyof assemblyin whichthe CD3-y,6, e chains forma core structure to whichTCR-~/flchains bind. At this point, an alternative modelin which the TCR-a/fl and CD3-y,6, ~ chains randomlyassociate in the mature T lymphocytecannot be ruled out. The TCR-a/fl--CD3-7, b, e Complex ls Formed Within the Endoplasmic Reticulum (Figure 5) Regardless of the hierarchy of assembly described above, pulse-chase labeling experiments have shownthat the CD3-v,6, e proteins form a complexwith the TCR-a/flheterodimerwhile residing in the endoplasmic reticulum. Theevidencefor this centers on the observationthat complete assemblytakes place before the N-linked oligosaccharides of the componentchains have beenmodifiedby enzymesknownto reside in the Golgi apparatus. Moreover,the addition of monensin,an inhibitor of Golgi functions, preventedthe maturation of the oligosaccharides attached to

Annual Reviews

Annu. Rev. Immunol. 1988.6:629-662. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

THE TCR/CD3

COMPLEX 643

the various polypeptidechains, but did not affect the TCR-CD3 assembly (58). Althoughthe assemblyof the TCRand CD3chains is a rapid process, the export of the complexfrom the ERto the Golgi is slow, requiring 3 hr for the processing of half the complexesfrom the Endo-H-sensitive to the Endo-H-resistantforms(58). This confirmsearlier results of Borst et al (26), whoshowedthat completesialylation of the CD3complex, modificationthat occursin the trans-elementsof the Golgi, is also a slow process. Thefactors that control the export of the complexfromthe ERto the Golgi stacks are unknown.The loosely associated CD3-(mayplay crucial role (59). However,it is apparent that association of TCR/CD3 not rate-limiting in driving export, since the assemblyof CD3-(with the rest of the complexis very rapid (B. Alarcon,unpublished). Recently, a newtype of immunedeficiency has been described that is characterized by the lack of TCR/CD3 expression on the surface of peri÷ and expressed normal levels of pheral T lymphocytes,whichwere CD2 CD4or CD8(60). The TCR/CD3 complex was assembled in the endoplasmic reticulum as in normalT cells, but the complexwas not exported to the Golgiapparatus(B. Alarcon,B. Regueiro,A. Arnaiz,andC. Terhorst; submitted). Althoughthe reasons for this export defect are unknown, naturally occurring variants such as these maybe helpful in elucidating the export mechanisms. Incompletely Assembled Receptor Complexes Do Not Reach the Plasma Membrane ManyT cell mutantsand T cell variants that lack one or morechains of the TCR/CD3 complexhave been described. In the eases analyzed to date, the proteins that are synthesizedassembleinto "subcomplexes," but they remain inside the cell. Transportof the complexto the plasmamembrane can be reconstituted by transfer of the cDNA encodingthe missing chain into the mutants. Similarly, in COScells, transfected CD3-y,-~, and -e assemblebut remain inside the cell. These observations are consistent with the notion that complete assembly of the TCR/CD3 complexis a prerequisite for export fromthe endoplasmicreticulum. Not all the newlysynthesized polypeptide chains appear to become incorporated into the receptor (58, 60a). Amajor question posedby these results is the site of degradation.As degradationof polypeptidechains does not occur in the endoplasmic reticulum and since the TCR/CD3 complexis formed in the endoplasmic reticulum, a transport pathway fromthis organelleto a lysosomal(sub-)compartment has to exist. Studies using T cell mutantsandvariants and COScell transfections showthat the N-linkedglycans of the chains of partial complexes remainunprocessed.It

Annual Reviews

Annu. Rev. Immunol. 1988.6:629-662. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

644

CLEVERS ET AL

seemslikely, therefore, that they movedirectly from the endoplasmic reticulum to the degradative compartmentvia a pathwaythat avoids the distal stacks of the Golgi. Site-directed mutagenesisexperiments that introduce charged amino acids into the transmembrane domainsof viral surface glycoproteinshavc resulted in retention of the protein inside the cell andinterestingly, rapid degradationof the mutantprotein within lysosomes(61, 62). It is possible that the charges within the transmembranedomains of the TCR/CD3 proteins(Figures2 and3) are responsiblefor their retentionin the cell prior to assemblyand mayeven direct unassembledchains and "subcomplexes" along a degradative pathway. Duringassembly, the negative charges of the CD3proteins maybe neutralized by the positive charges of the TCR. Regardlessof mechanism,a series of mutagenesisexperimentsdirected at the transmembraneregions of the TCR/CD3 proteins will define their particular role in assemblyand transport. Other retention signals may, however, exist in the polypeptide chains that composethe TCR/CD3 complex. CD3-o9 Plays a Role in Assembly (Figure 4) Pulse-chase biosynthetic labeling experimentsdetect a nonglycosylated polypeptidechain of 28 kd (CD3-~o)that associates with the CD3complex early during biosynthesis but does not travel with the receptor to the plasma membrane(63). CD3-~ocould be detected in humanperipheral lymphocytes,humanT leukemia cell lines (58, 63) and murine T-T hybridomas(B. Alarcon, unpublished). Morerecently, wehave providedevidencethat CD3-o9 also associates with single TCR-~ and -fl chains. Associations with CD3-~ooccur before the N-linked glycans of the receptor complexhave been processed and probably take place in the endoplasmic reticulum(58). Basedon partial N-terminalsequencedata and on the lack of cross-reactivity with any of the anti-TCRor -CD3reagents, CD3-~o appears to be distinct from the other membersof the complex(63 and B. Alarcon, unpublished). Whatrole does CD3-~oplay in assemblyand transport? The export of individual chains of the CD3/TCR complexand the fully assembledreceptor itself from the ERis slow. Giventhat approximatelyhalf of the membraneof the ERvesicularizes and movesto the Golgi every 10 min (64), it seemsprobablethat the polypeptidesof the receptor are actively retained within the ER.CD3-o9mayretain partially assembledcomplexes at the site of assemblyuntil the process is completed.In a comparable system, Ig heavy chains assemble with a protein termed BiP. Assembly takes place in the ER,displacing BiP fromthe Ig heavychain (64). Unfortunately, a retention modelcannot be completelyreconciled with

Annual Reviews

Annu. Rev. Immunol. 1988.6:629-662. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

THE TCR/CD3COMPLEX 645 the observedrapid degradationof individual chains and incompletereceptors. Suchobservationsrequire that the chains in question leave the ER. It is likely that retention and targeting for degradationare coupled.The CD3-~o chain maybe involved in the transport ofreceptor-"subcomplexes" from the ERto lysosomes. This notion is supported by the observation that the CD3-~,-6, and -~ complexesare stabilized by treatment with monensin(63). Pulse-chase biosynthesis experiments showthat CD3-o~ dissociates from the TCR/CD3 complexearly during biosynthesis. The mechanismsthat control this dissociation remain unknown.It could be that CD3-~is displacedduringthe final stages of assembly.Alternatively, CD3-~o could act as a catalyst of assembly,which, for instance, could be necessary to provide for a microenvironmentin the membranewhich wouldbe destabilized by the chargedresidues in the transmembrane segments. On the other hand, no CD3-o~has been detected in COScell transfectants. It is therefore possible that CD3-~0 maynot be necessaryfor the CD3-v,6, ~ core assembly. In conclusion, the role of this newlydetected CD3-~o protein could be in: (a) retention of unassembledsubcomplexesin the endoplasmicreticulum; (b) guiding unassembledchains and subcomplexestowards the degradativepathway:(c) facilitating assembly.

SIGNAL TRANSDUCTION TRIGGERED VIA THE TCR/CD3 COMPLEX Numerousstudies have madeit clear that occupancyof the TCR/CD3 complexresults in a series of early metabolicevents that lead to T cell activation (65). Whilethe cytoplasmic domainsof the TCRpolypeptide chains are extremelyshort, CD3polypeptidechains contain substantially longer intracellular domains(Figure 3) and are therefore likely to mediate signal transduction upon antigen recognition by the TCRheterodimer. Noneof the CD3genes cloned so far contains kinase domains or GTP binding sites, althoughthese havebeenfound in growthfactor receptors. Signal transduction through the TCR/CD3 protein ensemblewill therefore probably involve currently unidentified membraneor cytoplasmic proteins. Early changes observedafter triggering of the TCR/CD3 complexwith either antigen of MAb are: a rise in intracellular Ca2+, + a stimulated K efflux (66), increase of the turnover of phosphoinositides,activation protein kinase C, and a rise of the intracellular pHby activation of a ÷ antiporter. In addition, poorly defined secondary signals proNa+/H vided by accessorycells like macrophages act synergistically in the induc-

Annual Reviews

_646

CLEVERS ET AL

tion of T cell proliferation. This discussionis confinedto several early events in T cell activation via the TCR/CD3 pathwayand the role of phosphorylation of the TCR/CD3 complex.

Annu. Rev. Immunol. 1988.6:629-662. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

The Role of Ca2÷ and PhosphatidylInositol in T Cell Activation Historically, manyinvestigations haveshownthat Ca2+ ions play a crucial role in T cell activation. Theinductionof T cell proliferation andcytotoxic activity is blockedreversibly by Ca2÷ chelators. Severalgroupshaveshown that triggering of the TCR/CD3 complexof resting T cells, T cell clones, and T leukemiccell lines by MAbs,mitogenic lectins, or appropriately presentedantigen results in a rise in intracellular free Ca~÷ [(Ca2÷)i]. In the leukemiccell line Jurkat, an initial rise in (Ca2+)i is mediatedby the transient release of Ca2+ fromintracellular stores; a sustained elevation of (Ca2÷)ioccurs only if extracellular 2÷ isavailable. Similarly, in resting humanor murineT cell lines (64a) andin T cell clones, the major componentof the rise in (Ca2+)i is causedby an influx of Ca2+ from the extracellular milieu(see 65 for review). Recently, Kunoet al identified mitogen-dependentCa2÷ channels in humanT cells using a patch clamptechnique (69). The authors recorded openingof putative Ca2+ channels in cloned humanT helper cells after addition of PHAor anti-CD3 MAboutside the patch pipette. It was arguedthat the gigaohmseal of the patch pipette preventedthe entry of the inducingreagent into the patch area wherethe changeswererecorded, pointing to the requirement for an intermediary metabolic event. The nature of one such metabolicintermediateis discussed below. In a rapidly growingnumberof receptor activation models, Ca2+ mobilization results from ligand-inducedsignaling through the phosphatidyl inositol (PI) pathway. This bifurcated second messenger mechanism involvesthe hydrolysisof PI 4,5 bisphosphateinto the biologically active substances inositol 1,4,5 trisphosphate (IP3) and diacylglycerol (DAG) uponreceptor activation. IP 3 subsequentlyindues a rise in (Ca2+)i; DAG activates a key enzymein cellular activation, protein kinase C (67). Most of the biochemicalparametersassociated with ligand inducedPI hydrolysis have indeed been observed in T cells stimulated through the TCR/CD3 complex.The formation of IP3 has been measureduponactivation of the TCR/CD3 complexby MAb(65) and by antigen (68). Moreover, IP3 been shownto mediate the release of Ca2+ from saponin-permeabilized Jurkat cells (65), indicatingthat IP3 indeedmobilizesCa2+in T cells. This notion is corroborated by a recent study demonstratingthe presence of IP3-activated Ca2÷ channels in the T cell membrane(69). The direct involvementof protein kinase C in T cell activation is evidencedby its

Annual Reviews THE TCR/CD3

COMPLEX 647

translocation from cytosol to the membraneupon perturbation of the TCR/CD3complex (70). Given the observations described, it could be hypothesized that coupling of phospholipase C to the TCR/CD3 complex is mediated via G proteins. Someindirect evidence for the involvement of GTP-bindingproteins in T cell activation has recently been obtained (71). However,definitive proof for such a coupling will require more detailed biochemical studies.

Annu. Rev. Immunol. 1988.6:629-662. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

CD3 Phosphorylation

Accompanies

T Cell

Activation

HumanT lymphocytes exposed to phorbol esters, ionomycin, the mitogen PHA,or antigen and cultured in the presence of 32p phosphate contain a phosphorylated CD3-~chain. Someexperiments involving induction of a phosphorylated human CD3-~or CD3-e have been reported (72, 73). Further analysis of the CD3-), chain showedthat a single site (serine 126) was phosphorylated when T lymphocytes were stimulated by PHA or PMA.In contrast, two sites (serines 123 and 126) were phosphorylated in response to the Ca2+ ionophore ionomycin (72). Thus, these phosphorylation events may reflect the regulation of phosphorylation by two independent pathways. Manycell surface structures have the ability to recycle. In particular receptor moleculesthat deliver ligands to the cell, e.g. transferrin receptor or LDL-receptor, are endocytosed and reappear on the cell surface rather rapidly. It had been observed that antibodies directed at humanTCRor CD3are endowed with the ability to induce TCR/CD3internalization (74). Whenit was found that phorbolesters similarly could induce internalization, a careful study of the relationship between phosphorylation of the TCR/CD3complex and endocytosis was conducted by Krangel (75). Phosphorylated forms of the CD3-~ protein were shown to be constitutively endocytosed, while nonphosphorylated forms of the molecule were excluded from that pathway. Similar results were obtained in mouse T lymphocytes (37, 38). It thus appears that the phosphorylation of CD3), mediates a down regulation of the TCR/CD3complex. Howthis down regulation is coordinated with antigen-specific T cell activation needs to be resolved. In a series of studies, Samelsonet al have provided strong evidence for antigen-induction of tyrosine phosphorylation of a polypeptide (CD3-p21) associated with the antigen receptor on murine T cells (40, 41, 76). The isolated TCR/CD3complex does not appear to have tyrosine kinase activity and must therefore be coupled to an as-yet-unknown kinase. In contrast to the antigen-induced tyrosine phosphorylation in helper T cell lines, T cells of some immunodeficiency mice (gld and lpr) contain constitutively tyrosine phosphorylated CD3-p21(41). Since the lpr and

Annual Reviews

648

CLEVERS ET AL

Annu. Rev. Immunol. 1988.6:629-662. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

gld T cells are inert in termsof their responsesto antigenor lectins, it is possible that the CD3-p21 phosphorylationcauses desensitization of the TCR/CD3 complexresulting in a decreasedefficacy of signal transduction. The disulfide linkage of CD3-p21and CD3-(maybe a prerequisite for phosphorylation of CD3-p21.Thus, both CD3-p21and CD3-( mayturn out to play a pivotal role in the TCR/CD3 transduction mechanism.

Interactions Between the TCR/CD3- and Antigen-Independent Activation Pathways All of the biochemical changes accompanyingTCR/CD3 triggering occur in purified T cell populations or in single T lymphocytes.However,the proliferative response to anti-TCR/CD3 reagents and the production of the lymphokinesI1-2 and y-interferon require the presence ofmacrophages capable of interacting with the activating MAbthrough their surface Ig receptors. In an attempt to circumvent the macrophage-dependencyof T cell proliferation, a numberof manipulationshavebeendefined that, in concert with anti TCR/CD3 monoclonalantibodies, result in T cell proliferation and lymphokineproduction. Somegroups have observed that the monokineIL- 1 can functionas an additionalsignal for T cell activation, although this could not be confirmedby others. Furthermore,the phorbol ester PMA, an irreversible activator of protein kinase C, at low concentrations synergizes strongly with anti-TCR/CD3 Mabsin the induction of T cell proliferation (65). However,despite the studies described above and numerous others on this subject, the nature of the elusive secondarysignal is not understood. Several antigens like CD2, CD28,CD4and CD8and Thy-1 have been identified that, upontriggering with Mabssynergize with anti-TCR/CD3 monoclonalantibodies to induce functional responses in T cells. Antibodies to the adhesionmoleculeCD2specifically block T cell functions. Different CD2epitopes have been defined that either block (CD2-1) activate (CD2-2 plus 3) T cell proliferation or killer function (92, 93). does the CD3pathway,activation ofT cells via CD2utilizes the hydrolysis of PI phosphatesfor its signal transduction (94). The CD2pathwayproceeds normally in CD3-negativeNKcells (95). However,anti-CD3Mabs block the CD2pathway in thymocytes (96). Also, modulation of the TCR/CD3 complexfrom cloned T cells abolishes their response to antiCD2Mabs.It has been hypothesizedthat the interaction betweenthe CD2 and the TCR/CD3 pathwaysaccounts for the elimination of autoreactive cells in the thymus. In this modelit is proposedthat thymocytesare

Annual Reviews

Annu. Rev. Immunol. 1988.6:629-662. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

THE TCR/CD3 COMPLEX

649

activated to grow via the CD2pathway. Whenever the TCR/CD3complex is triggered simultaneously by autoantigens, the growth of such thymocytes is blocked and they eventually die (97). Similarly a large body of data supports the notion that CD4and CD8 act in concert with the TCRto optimize responses to TCR-antigeninteractions. Both transfection studies (98-101) and experiments with monoclonal antibodies (15a) support the idea that the signal transduction pathways of TCR/CD3and CD4synergize. Suggestions that TCRs can form transient complexes on the cell surface with CD4have not yet been supported by biochemical studies. CD28,initially identified by the Moab9.3, is expressed on a subset of matureT cells (102). The 9.3 antibody is mitogenicfor resting T cells in the presence of macrophages or the phorbol ester PMA.Like the TCR/CD3 complex, CD28possibly signals through the PI pathway, since crosslinking of this antigen on the T cell surface mobilizes Ca2+ (103). Moretta et al recently isolated variants of Jurkat cells that lacked CD3but expressed CD28.These variant cells failed to produce IL-2 after triggering with 9.3 (104), indicating that activation via CD28requires the presence of the TCR/CD3 complex. The isolated murine Thy-l.2 gene has b~en transfected into Jurkat cells (a humanT leukemic cell line) and into mudneB cell lines (105). Transfectant cells that expressed the Thy-1.2 antigen demonstrateda rapid rise of intracellular Ca2+ upon addition of anti-Thy-1 antibodies. This could also be done in the B lymphomacell lines. The induction of a rise in intracellular Ca2+ was therefore thought to be independent of the presence of the TCR/CD3complex on the surface of Thy-1 expressing cells. Mutant Jurkat cell lines that lack surface expression of TCR/CD3 and their revertants were recently used for transfection of the Thy-l.2 gene + Jurkat cells responded to anti-Thy-1 antibodies (106). Normal TCR/CD3 with a rise in (Ca2+)i and, in the presence of phorbolesters, with IL-2 secretion. TCR/CD3variants responded to anti-Thy antibodies with the rise in Ca+ + but did not secrete IL-2 in the presence of phorbolester. The ÷ "revertants" anti-Thyl-induced IL-2 secretion was restored in TCR/CD3 transfected with the TCRa or fl cDNA (106). Similar results were obtained by Schmitt-Verhulst and colleagues (107) whoselected variants of a mufine CTLclone that had lost expression of the TCRa chain mRNA and which expressed normal amounts of Thy-1 on its surface. Antibodies to Thy-1 did not cause activation as measured by interferon y secretion. These data demonstrate unequivocally that Thy-1 mediated T-cell activation, as measuredby IL-2 or interferon- 7 production, requires coexpression of the TCR/CD3 complex.

Annual Reviews

650 THE CD3

CLEVERS ET AL

STRUCTURE GENES

Annu. Rev. Immunol. 1988.6:629-662. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

Expression of the Thymic Maturation

AND

REGULATION

OF

THE

TCR/CD3 Genes During

The T lineage pathway of differentiation represents one of a few such pathways that are initiated during fetal development and persist in the adult animal. Bone marrow-derived prothymocytes go through several stages of differentiation in the thymus to give rise to immunocompetent T lymphocytes. The mechanisms governing this process are incompletely understood, but center around the generation of diversity of TCR-specificity followed by the selection of a limited numberof mature T cells to be added to the immunesystem (see 1 and 77 for review). During fetal development in the mousethe first detectable population of thymocytes is a CD4-, CD8-("double-negative") subset at day 14 gestation (78). At days 16 and 17, double-positive and mature singlepositive T cells begin to be detectable and increase in proportion. A distribution of the different thymocyte-subsetssimilar to that found in the adult thymus is reached by day 19 of fetal life. To demonstrate putative precursor/progeny relationships, experiments of adoptive transfer, organ culture, and culture of isolated cells have been performed. The doublenegative subset has been demonstrated to generate all the other subsets (79, 80). On the contrary, it has not been possible to generate singlepositive mature thymocytes from the double-positive subset. Whether this subset represents an intermediary stage or an end point in T cell differentiation (see Figure 6) is still controversial. The T cell receptor genes are rearranged and expressed sequentially during maturation in both thymic ontogeny and adult thymus (see 1 and 77 for review). The first gene to be rearranged is TCR-),at day 14 ofmurine fetal development (81), followed by TCR-fl gene rearrangement. TCR-~ gene rearrangements are first detected at day 16. Levels of detectable transcripts of the TCRgenes parallel the occurrence of their respective rearrangements. Appearance of the TCR/CD3complex on the surface of thymocytes requires transcription of the genes of all of its members.In two exhaustive studies of normal thymocytesas well as of a series of humanleukemias it was reported that all cells analyzed expressed CD3-transcripts and CD3proteins (82-84, 91). Thymocytes or leukemic cells that contain TCRtranscripts do express the TCR/CD3-complex on the cell surface, whereas the ones lacking at least TCR-~mRNA accumulate CD3-y, T3-6, and T3e chains intracellularly, in the perinuclear envelope. In addition, thymocytes of the double-negative subset lack TCR-fl transcripts yet have

Annu. Rev. Immunol. 1988.6:629-662. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

Annual Reviews THE TCR/CD3 COMPLEX 651

Annual Reviews

Annu. Rev. Immunol. 1988.6:629-662. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

652

CLEVERS ET AL

intracellular CD3proteins (83). Thetranscription and translation of the CD3genes must therefore be a very early phenomenon in the maturation of thymocytes.It is at present unknown whetherthe transcription of CD3 genesis initiated before or after enteringthe thymus. A small percentage of double-negativethymocytes(0.2%4).9%)express the CD3complexon the cell surface. This population does not have TCR~ nor TCR-flmessagesbut does express TCR-~and TCR-t5gene products. Thepresence of TCR-~/~iheterodimersprecedes that of TCR-~/flheterodimers during mousethymic ontogeny. TCR-~/6-and TCR-~/fl-expressing T cells presumablyconstitute different lineages since TCR-~/6-expressing cells have been demonstratedin peripheral organs and appear to be functionally mature(20, 22, 23, 49) (see Figure The CD3 Genes Asa first step towardsthe understandingof the coordinate expressionof the CD3chains during T lymphocytematuration, a detailed analysis of the structure and the regulation of the genes encodingthe CD3-~,,CD3-6, and CD3-echains has been initiated. All three genes were originally mapped to band 11q23 in human and to mouse chromosome9 (8587). Morerecently pulsed-field DNA electrophoresis has been applied to demonstratethat the CD3genes are clustered within a 300 kd-region on humanchromosome 11 (87; H. Clevers, S. Dunlap, H. Saito, T. Wileman, C. Terhorst). The genes for CD3-?and CD3-6are closely linked in both mouseand human;the genes are separated by approximately1.5 kb and organized in a head-to-headconfiguration (87, 88). The CD3-6gene is split into five exons; the CD3-~gene contains 7 exons. Exon-intron boundaries are located at homologous positions in the two genes with the exception that the CD3-~:gene contains an additional 24 bp coding exon (exon 2) and separate 3’ untranslatedexon(see Figure7A).Giventhis similarity of gene organization, physical linkage, and the high sequencehomologyof the CD3-yand CD3-6genes, it is highly likely that these two genes arose througha duplication event. A very close linkage of geneswith divergent transcription is rare, andin several of those instances regulatoryelements do exist in the area betweenthe transcriptionstart sites (88). The CD3-egene consists of eight exons two of which are unusually small, encoding6 and 5 aminoacids of the N terminusof the protein (see Figure 7B). The overall homologybetween CD3-eand the CD3-~/CD3-6 genepair is relatively low, beingconfinedto areas aroundthe Cys-residues in the extracellular domainsof the chains. Uponcomparisonof the genomic sequences the short stretch of high homologylocated directly Nterminal of the transmembranesegment (53, 55) was found to coincide

Annu. Rev. Immunol. 1988.6:629-662. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

Annual Reviews THE TCR/CD3 COMPLEX 653

Annual Reviews 654

CLEVERS ET AL

5 ~ UT

LP

EX

TM I~

~’ UT

mRNA

GENE ~

Annu. Rev. Immunol. 1988.6:629-662. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

tkb Figure 7B The murine CD3-~gene (H. Clevers, S. Dunlap, H. Saito, T. Wileman, and C.

Terhorst,submitted).UT,untranslated;LP,leaderpeptide;EX,extracellular;TM,transmembrane; IN,intracellular. with the presence of a conserved boundary (H. Clevers, S. Dunlap, H. Saito, T. Wileman,and C. Terhorst, submitted). This observation strongly suggests that all genes in the CD3gene cluster are derived from a common ancestral gene. A series of sequence comparisons suggests an evolutionary relationship between the various CD3sequences and the Ig superfamily. Amongthe Ig-superfamily sequences, the CD3sequences (in particular CD3-e) may fit best with the N-CAM domains, at about the same level of significance as those seen between fl2-microglobulin and IgG domains (54, 108). Transcription of eukaryotic genes is mediated by control regions composed of complex combinations of promoter and enhancer elements (cisacting elements), arrayed in tandem, that allow multiple distinct transacting factors to regulate RNAsynthesis. This mosaic arrangement of regulatory sequences provides genes with the possibility of using sets of commonelements. The unique expression pattern of any given gene is obtained through the composition and spatial organization of such commonelements, acting in concert with elements that bind inducible or tissuespecific factors. Based on the evolutionary relationship and on the coordinate expression of the CD3genes, the T cell-specificity of these genesis likely to be governed by overlapping sets of regulatory factors. This notion is corroborated by the recent characterization of the T cell mutant HY-827-P19. This cell line expresses the TCR-aand TCR-fl chains but, unlike its parent cell line, lacks all of the CD3polypeptide chains (B. Alarcon, in preparation). Since in this mutant all CD3genes are silenced simultaneously, it is most likely that not the CD3 genes themselves but the gene encoding a common regulatory factor has been damaged. Moreover,this cell line demonstrates that the expression of the CD3genes can be uncoupled from TCRgene expression.

Annual Reviews THETCR/CD3COMPLEX 655 Table2 Chromosome location of T cell receptor/CD3 genes

Annu. Rev. Immunol. 1988.6:629-662. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

Human chromosome TCR ~ /3 y 3

14 q 11 7 q 32-35 7p 15 --

CD3 ~ 6 ~

11 q23 11 q23 11 q 23

Mouse chromosome

14 6 13 14

C-D B A 2-3 C-D

9 9 9

All CD3genes are transcribed from non-TATApromoters (56a, 87-89, H. Clevers, S. Dunlap, H. Saito, T. Wileman, C. Terhorst, submitted). Because of the close proximity of the divergently transcribing CD3-yand CD3-3promoters, single regulatory factors acting on the intervening gene segmentpotentially affect transcription of both genes. In vitro gene regulation analysis has revealed the presence of a T cell-specific enhancer element directly 3’ of the CD3-3gene. The location of this enhancer element coincides with the presence of a T cell-specific DNaseI hypersensitive site. Interestingly, the sequenceof this element is repeated in the CD3-y/CD3-6intergenic segment (K. Georgopoulos, P. Van den Elsen, E. Bier, A. Maxam,and C. Terhorst, submitted). Furthermore, a similar motif is present directly upstream from the CD3-e gene promoter. Sequence comparison of the region upstream of the CD3-egene with the CD3-y/CD3-6intergenic region revealed the presence of another homologous motif. These two motifs are excellent candidates for T cell-specific cis-acting elements. While the three knownCD3genes are closely linked to each other, no linkage exists with the TCRgenes (Table 2). CONCLUSION Despite rapid progress in the molecular anatomy of the TCR/CD3 complex, muchneeds to be learned about its function. Several new polypeptide chains (in addition to CD3-p21, CD4and CD8)will probably found coupled to this dynamic protein ensemble. Our current knowledge would predict that they are either G-proteins or tyrosine kinases. Yet, completely different pathways maybe found. A rapidly increasing number of functional and structural mutants of T cell lines combinedwith transfection of normal and modified genes coding for all the membersof the

Annual Reviews

Annu. Rev. Immunol. 1988.6:629-662. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

656

CLEVERS ET AL

ensemble will facilitate these new discoveries. However, even if the molecular interactions of the TCR/CD3 complexare workedout, the synergism with other T cell surface structures in the induction of T lymphocyte proliferation needs to be understood. Future studies of assembly of the TCR/CD3 complex will answer major questions about retention in the endoplasmicreticulum, the existence of a hitherto unknownintracellular transport pathway, and the regulation of the level of cell surface expression during thymic differentiation. Future studies of the coordinate regulation of transcription of the TCRand of the CD3genes will be of interest as a model of T cell-specific gene expression. Thus, principles of signal transduction by the TCR/CD3 complex, of its assembly,and of the regulation of its genes will not only be of great interest to T cell biologists but will have general implications in the fields of developmental and cell biology.

ACKNOWLEDGMENTS

The authors wish to thank Drs. C. Hall and J. Bergelson for critically reading the manuscript; Drs. L. Samelson, A. Weiss, J. Coligan, D. Pardoll, and A. Kruisbeek for sharing experimental data; and J. W. Lockhart for expert secretarial assistance. H. Clevers is supported by a fellowship from the Dutch Cancer Society (KWF),B. Alarcon is supported by a fellowship from E.M.B.O., T. Wilemanis a fellow of the Puritan Bennett Foundation. These studies were supported by NIHgrants AI-15066 and AI-17651, and ACS grant No. IM-289E.

Literature Cited 1. Allison, J. P., Lanier, L. L. 1987. The structure, function, and serology of the T cell antigen receptor complex. ~4nn. Rev. Immunol. 5:503-40 2. Toyonaga, B., Mak, T. 1987. Genes of the T cell antigen receptor in normal and malignant T cells. Ann. Rev. Immunol. 5:585~20 3. Kronenberg, M., Siu, G., Hood, L. E., Shastri, N. 1986. The molecular genetics of the T cell antigen receptor and T cell antigen recognition. Ann. Rev. Immunol. 4:529-41 4. Spits, H., Schooten, W. V., Keizer, H., Seventer, G. V., Van de Rijn, M., Terhorst, C,, De Vries, J. E. 1986. Alloantigen recognition is preceded by a non-specific adhesion of cytotoxic T

cells and target cells. Science 232: 4035 5. Yague, J., White, J., Coleclough, C., Kappler, J., Palmer, E., Marrack, P. 1985. The T cell receptor: The ct and fl chains define idiotype, and antigen and MHC specificity. Cell 42:81 6. Dembic, Z., Haas, W., Weiss, S., McCubrey, J., Kiefer, H., yon Boehmer, H., Steinmetz, M. 1986. Transfer of specificity by murine ~ and /3 T cell receptor genes. Nature 320:232 7. Brenner, M. B., McLean,J., Dialynas, D. P., Strominger, J. L., Smith, J. A., Owen,F. L., Seidman, J. G., Ip, S., Rosen, F., Krangel, M. S. 1986. Identification of a putative second T cell receptor. Nature 322:145-49

Annual Reviews

Annu. Rev. Immunol. 1988.6:629-662. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

THE TCR/CD3 COMPLEX657 8. Borst, J., van de Griend, R. J., van Oostveen,.I.W., Ang,S. L., Melief,C. J., Seidman,J. G., Bolhuis, R. L. H. 1987. TheT cell receptor ~/CD3complex found on cloned functional lymphocytes. Nature 325:683-87 9. Alarcon,B., Pettey, C., Boylston,A., Yssel, H., DeVries, J., Terhorst, C., Spits, H. 1987.A humanNKlike T cell clone uses the T cell receptor y/CD3 complexin cytolysis. Proc.Natl. Acad. Sci. USA84:3861~55 10. Kappler, J. W., Roehm,N., Marrack, P. 1987.T cell toleranceby clonalelimination in the thymus.Cell 49:273-80 11. Kappler, J. W., Wade,T., White, J., Kushnir, E., Blackman,M., Bill, J., Roehm,N., Marrack,P. 1987. A T cell receptor V/~segmentthat impartsreactivity to a class II major histocompatibility complexproduct. Cell 49: 263-71 12. Matis,L. A., Sorger,S. B., McElligott, D.L., Fink,P. Z., Hedrick,S. M.1987. Themolecularbasis of alloreactivity in antigen-specific, major histocompatibility complex-restricted T cell clones. Cell 51:5(~69 13. Springer, T. A., Dustin, M.L., Kishimoto, T. K., Marlin, S. 1987. The lymphocytesfunction associated LFA1, CD2, and LFA-3molecules: cell adhesion receptors of the immune system. Ann. Rev. Immunol.5:223-52 14. Littman,D. 1987. Thestructure of the CD4and CD8genes. Ann. Rev. Immunol. 5:561 15. Emmerich,F., Strittmatter, U., Eichmann,K. 1986. Synergismin the activation of humanCD8T cells by crosslinking the T cell receptor complex with the CD8differentiation antigen. Proc. Natl. Acad. Sci. USA83:8298-8302 15a. Saizawa,K., Rojo,J., Janeway,C. A. Jr. 1987.Evidencefor a physicalassociation of CD4andthe CD3: ~t:/~: T cell receptor. Nature 328:260-63 16. Meuer,S. C., Cooper,D. A., Hodgdon, J. C., Hussey,R. E., Fitzgerald, K.A., Schlossman, S. F., Reinherz, E. L. 1983. Identification of the antigen/ MHC-receptor on human inducer T lymphocytes.Science 222:1239 17. Haskins, K., Kubo, R., White, J., Pigeon, M., Kappler, J., Marrack,P. 1983. The major histocompatibility complex-restrictedantigen receptor on T cells, i. Isolation with a monoelonal antibody. J. Exp. Med.157: 1149~i9 18. Hedrick,S. M., Cohen,D. I., Nielson, E. A., Davis, M. 1984. Isolation of eDNAclones encodingT cell-specific membrane-associated proteins. Nature

308:149 19. Yanagi,Y., Yoshikai,Y., Leggett,K., Clark, S. P., Aleksander,I., Mak,T. W. 1984. A humanT cell-specific cDNA clone encodesa protein having extensive homology to immunoglobulin chains. Nature 308:145 20. Lanier, L., Weiss, A. 1986. Presence of Ti (WT31)negative lymphocytes normalblood and thymus.Nature324: 268-70 21. Krangel,M. S., Bierer, B. E., Devlin, P., Clabby, M., Strominger, J. L., McLean,J., Brenner, M. B. 1987. T3 glycoprotein is functional although structurally distinct on humanT cell receptorSci. ),/TUSA lymphocytes. Proc. Natl. ~4cad. 84:3817-21 22. Lew,A. M., Pardoll, D. M., Maloy,W. L., Fowlkes, B. J., Kruisbeek, Cheng,S. F., Germain,R. N., Bluestone, J. A., Schwartz,R. K., Coligan, J. E. 1986. Characterizationof T cell receptor gamma chain expression in a subset of murine thymocytes.Science 234:1401-5 23. Pardoll, D. M., Fowlkes,B. J., Bluestone, J. A., Kruisbeek,A., Maloy,W. L., Coligan,J. E., Schwartz,R. 1987. Differentialexpression.of twodistinct T cell receptors during thymocyte development.Nature 326:79-81 24. van Agthoven,A., Terhorst, C., Reinherz, C., Sehlossman, S. F. 1981.Characterization of T cell surface glycoproteins T1 and T3 present on all humanperipheral T lymphocytes and functionally mature thymocytes.Eur. J. Immunol.11:18-21 25. Borst, J., Prendiville, M.A., Terhorst, C. 1982. Complexityof the humanT lymphocyte-specific cell surfaceantigen T3. J. Immunol.128:1560 26. Borst, J., Alexander, S., Elder, J., Terhorst, C. 1983. The T3 complex on humanT lymphocytes involves four structurally distinct glycoproteins.J. Biol. Chem.259:5135 27. Kanellopoulos, J. M., Wigglesworth, N. M., Owen, M. J., Crumpton, M. J. 1983. Biosynthesis and molecular nature of the T3 antigen of humanT lymphocytes. EMBO J. 2:1807 28. Bor~t,J., Prendiville,M.A., Terhorst, C. 1983. The T3 complex on human thymus-derived lymphocytescontains twodifferent subunitsof 20 k Da. Eur. J. Immunol.13:576 29. Borst, J., Colligan,J. E., Oettgen,H., Pessano, S., Malin, R., Terhorst, C. 1984. The6 and e chains of the human T3fr cell receptor complexare distinct polypeptides. Nature312:455

Annual Reviews

Annu. Rev. Immunol. 1988.6:629-662. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

658

CLEVERSET AL

30. Pessano,S., Oettgen,H., Bhan,A. K., Terhorst, C. 1985.TheT3/Tcell receptor complex: antigenic distinction betweenthe 20-kd T3 (T3-6 and T3-e) subunits. EMBO J. 4:337 31. Meuer, S. C., Fitzgerald, K. A., Hussey,R. E., Hodgdon, J. C., Schlossman,S. F., Reinherz, E. 1983. Clonotypie structures involvedin antigenspecific human T cell function:relationship to the T3 molecularcomplex.J. Exp. Med. 157:705 32. Oettgen,H., Kappler,J., Tax,W.J. M., Terhorst, C. 1984. Characterizationof the two heavychains of the T3 complex on the surface of the humanT lymphocytes. J. Biol. Chem.259:12039 33. Meuer,S. C., Acuto, O., Hussey, R. E., Hodgdon, J. C., Fitzgerald, K. A., Schlossman, S. F., Reinherz, E. L. 1983. Evidencefor the T3-associated 90KD heterodimeras the T cell antigen receptor. Nature303:808 34. Zanders,E. D., Lamb,J. R., Feldman, M., Green,N., Beverly,P. C. L. 1983. Toleranceof T cell clonesis associated with membrane antigen changes. Nature 303:625 35. Ohashi,P., Mak,T., van denElsen, P., Yanagi,Y., Yoshikai, Y., Calman,A., Terhorst,C., Stobo,J., Weiss,A.1985. Reconstitution of an active surface T3/T cell antigen receptor by DNA transfer. Nature316:606 36. Saito, T., Weiss, A., Miller, J., Norcross,M. A., Germain,R. N. 1987. Specificantigen-Iaactivation of transfected humanT cells expressingmurine Ti ct t-humanT3 receptor complexes. Nature 325:125 37. Samelson, L. E., Harford, J. B., Klausner,R. D. 1985.Identification of the componentsof the routine T cell antigen receptor complex.Cell 43:223 38. Oettgen, FI., Pettey, C., Maloy,W., Terhorst, C. 1986. A T3-1ike protein complexassociated with an antigen receptor on murineT cells. Nature320: 272-75 38a. Berkhout,B., Alareon, B., Terhorst, C. 1987. Transfection of genes encoding the T cell receptor associated CD3complexinto COScells results in assemblyof the macromolecular structure. J. Biol. Chem.In press 39. Weissman,A. M., Samelson, L. E., Klausner, R. D. 1986. A newsubunit of the humanT-cell antigen receptor complex. Nature 324:480-82 40. Samelson,L. E., Maitray,P. D., Weissman,A. M., Harford, J. B., Klausner, R. D. 1986. Antigen activation of murineT cells induces tyrosine phos-

phorylationof a polypeptideassociated with the T cell antigen receptor. Cell 46:1083 41. Samelson, L. E., Davidson, W. F., Morse,H. C. III, Klausner,R. D. 1986. Abnormaltyrosine phosphorylation on T-cell receptor in lymphoproliferative disorders. Nature324:674 42. Novotny,J., Tonegawa,S., Saito, H., Kranz, D. M., Eisen, H. N. 1986. Secondary,tertiary, andquaternarystructure of T cell specific immunoglobulinlike polypeptide chains. Proc. Natl. Acad. Sci. USA83:742-46 43. Saito, H., Kranz,D. M., Takagaki,Y., Hayday, A, C., Eisen, H. N., Tonegawa,S. 1984. Completeprimarystructure of a heterodimericT-cell receptor deduced from cDNAsequences. Nature 309:757 44. Chien,Y. H., Becker,D. M., Lindsten, T., Okamura,M., Cohen,D. I., Davis, M.1984. A third type of murineT-cell receptor gene. Nature312:31 45. Murre, C., Waldmann, R. A., Morton, C. C., Bongiovanni,D. F., Waldman, T. A., Shows,T. B., Seidman,J. G. 1985. Human ?-chain genes are rearranged in leukaemic T cells and map to the short arm of chromosome 7. Nature 316:549 46. Brenner,M.B., McLean, J., Schejt, K., Riberdy,J., Ang,S. L., Seidman, J. G., Devlin, P., Krangel, M. S. 1987. Two formsof the T cell receptor ~ protein foundon peripheral blood cytotoxic T lymphocytes. Nature 325:689-94 47. Littman, D. R., Newton, M., Crommie,D., Ang, S.-L., Seidman, J. G., Gettner, S. N., Weiss, A. 1987.Characterizationof an expressed CD3-associated Ti ~,-chain reveals C? domainpolymorphism.Nature 326: 85-88 48. Moingeon, P., Jitsukawa,S., Faure, F., Troalen,F., Triebel, F., Graziani, M., Forestier, F., Bellet, D., Bohmon, C., Hercend,T. 1987. A 7-chain complex formsa functional receptor on cloned humanlympt~oeyteswith natural killer-like activity. Nature325:723-26 48a. Biron,C., vandenElsen, P., Tutt, M., Medveczky,P., Kumar,V,., Terhorst, C. 1987.T cell receptor geneexpression by Murinenatural killer cells and T cells stimulatedIn Vivo.J. Immunol.In press 49. Pardoll, D. M., Kruisbeek, A. M., Fowlkes,B. J., Coligan,J., Schwartz, R. H. 1987. Theunfolding story of T cell receptor ~,. FEBSJ.1:103 50. Chien, Y., Iwashima,M., Kaplan, K., Elliott, A. J. F., Davis,M. 1987.Anew

Annual Reviews

Annu. Rev. Immunol. 1988.6:629-662. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

THE TCR/CD3 COMPLEX

659

andsingleT cell receptor~t or fl chains. T-cell receptor genelocated withinthe alpha locus and expressed early in J. Biol. Chem.In press 59. McCleod,C., Mining, L., Gold, D., differentiation. Nature327:677 51. vandenElsen,P., Shepley,B. A., Bo(st, Terhorst, C., Wilkinson, M. 1986. Negativetransregulationof T-cell antiJ., Coligan,J., Markham, A. F., Orkin, gen receptor/T3 complex mRNA exS., Terhorst, C. 1984. Isolation of pression in murine T-lymphoma somaeDNAclones encoding the 20K T3 tic cell hybrids. Proc.Natl. Acad.Sci. glycoprotein of humanT-cell receptor complex. Nature 312:413-18 USA83:6989-93 60. Regueiro,J. R., Arnaiz-Villera, Ortiz 52. vanden Elsen, P., Shepley,B. A., Cho, M., Terhorst, C. 1985. Isolation and de Landazuri,M., Martin-Villa,J. M., characterization of a eDNAclone Vicario, J. L., Pascual-Ruiz,V., Guerencodingthe murine homologue of the ra-Garcia,F., Alcarni, J., Lopez-Botet, human20KT3/T-cell receptor glycoM., Manzanares, J. 1986. Familial protein. Nature314! 542-44 defect ofCD3(T3) expressionby T cells 53. Gold,D., Puck,J., Pettey, C., Cho,M., associatedwith rare gut epithelial cell Coligan, J., Woody,J., Terhorst, C. autoantibodies. Lancet 1:1274-75 1986. Isolation of eDNAclones en60a. Minami,Y., Weissman,A. M., Samelcoding the 20 kD non-glycosylated son, L. E., Klausner,R. D. 1987.Buildpolypeptide chains (T3-e) of the huing a multichain receptor: Synthesis, man T cell receptor/T3 complex. degradation,and assemblyof the T-cell antigenreceptor.Proc.Natl. Acad.Sci. Nature321: 431-34 54. Gold,D. P., Clevers, H., Alarcon,B., USA 84:2688-92 Dunlap,S., Novotny,J., Williams,A., 61. Davis, G. L., Hunter, E. 1987. A Terhorst, C. 1987. Characterization chargedaminoacid substitution within and expression of the murine T3-e the transmembraneanchor of the Roux chain: relationship to the immunoSarcomavirus envelope glycoprotein globulin supergenefamily. Proc. Natl. affects surfaceexpressionbut not intraAcad.Sci. USA.In press cellular transport. J. Cell. Biol. 105: 55. Krissansen,G. W., Owen,M.J., Verbi, 1191-1203 W., Crumpton,M. J. 1986. Primary 62. Adams,G. A., Rose, J. K. 1985. Incorstructure of the T3ysubunitof the T3/T poration of a charged aminoacid into cell antigen receptor complexdeduced the membranespanning domainblock from eDNA sequences:evolution of the cell surface but not membraneanT3Vand 6 subunits. EMBO J. 5: 1799choringof a viral protein. Mol.Cell. 1808 Biol. 5:1442-48 56. Krissansen,G. W., Owen,M. J., Fink, 63. Pettey, C. L., Alarcon,B., Malin,R., P. J., Crumpton,M.J. 1987. Molecular Weinberg,K., Terhorst, C. 1987. T3cloning of the cDNA encodingthe T3-V p28 is a protein associated with the 6 subunit of the mouseT31Tcell antigen and e chains of the T cell receptor-T3 receptor complex. J. lmmunol.138: antigen complexduringbiosynthesis.J. 3513-18 Biol. Chem.262:4854-59 56a. Tunnacliffe,A., Sims,J. E., Rabbitts, 64. Wieland,F. T., Gleason,M.L., SeraT. K. 1986. T3-6 pre-mRNA is transfini, M., Rothman, J. 1987.Therate of cribed from a non-TATApromoter bulk flow fromthe endoplasmicreticand is alternatively spliced in human ulumto the cell surface. Cell 50: 289cells. EMBO J. 5:1245-52 300 56b. Aruffo, A., Seed, B. 1987. Molecular 64a. Oettgen,H. C., Terhorst, C., Cantley, cloning of the CD2antigen, the T-cell L. C., Rosoff, P. M. 1985. Stimulaerythrocyte receptor, by a rapid imtion of the T3-T cell receptor communoselectionprocedure. Proc. Natl. plex induces a membrane-potentialAcad. Sci. USA84:3365-69 sensitive calciuminflux. Cell 40: 58357. Weiss,A., Stobo, J. D. 1984. Require90 mentfor the coexpressionof T3 and T 65. Weiss, A., Imboden,J., Hardy, K., cell antigen receptor on a malignant Manger,B., Terhorst, C., Stobo, J. humanT cell. J. Exp. Med.160: 12841986.Therole of the T3/antigenrecep99 tor complexin T cell activation. Ann. 58. Alarcon,B., Berkhout,B., Breitmeyer, Re~. Immunol.4:593-619 J., Terhorst, C. 1987.Assembly of the 66. Matteson, D. R., Deutsh, C. 1984. K humanT cell receptor-CD3 complex channels in T lymphocytes: a patch takes place in the endoplasmicreticclamp study using monocldnalantiulum and involves intermediary combody adhesion. Nature 307:468-71 plexes betweenthe CD3-(),, 6, e) core 67. Nishizuka,Y. 1984.Therole of protein

Annual Reviews

Annu. Rev. Immunol. 1988.6:629-662. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

660

CLEVERSET AL

kinase C in cell surfac~signal transduction and tumor promotion. Nature 308:693-98 68. Imboden,J., Weyand,C., Goronzy,J. 1987. Antigenrecognition by a human T cell cloneleads to increasesin inositol trisphosphate. J. Immunol.138: 132224 69. Kuno,M., Gardner,P. 1987. Ion channels activated by inositol 1,4,5-triphosphate in plasma membraneof human T-lymphocytes. Nature 326: 301-304 70. Nd, A. E., Bouic,P., Lattanze, G. R., Stevenson,H. C., Miller, P., Dirienzo, W., Stefanini, G. F., Galbraith, R. M. 1987. Reaction of T lymphocyteswith anti-T3 induces translocation of Ckinase activity to the membraneand specific substrate phosphorylation,d. Immunol:138:3519-24 71. Imboden,J. B., Shoback,D. M., Pardson, G., Stobo, J. D. 1986. Cholera toxin inhibits the T cell antigenreceptor-mediatedincreasesin inositol trisphosphate and cytoplasmic free calcium. Proc. Natl. Acad. Sci. USA 83:5673-77 72. Davies, A. A, Cantrell, D. A., Hexham, J. M., Parker, P. J., Rothbard, J., Crumpton, M. J. 1987. The human T3 ), chain is phosphorylated at serine 126 in responseto T lymphocyte activation. J. Biol. Chem.262:1 73. Cantrell, D., Davies, A. A., Londei, M., Feldman, M., Crumpton, M. J. 1987. Association of phosphorylation of the T3 antigen with immuneactivation of T lymphocytes.Nature 325: 54O 74. Meuer, S., Acuto, O., Hercend, T., Schlossman,S., Reinherz,E. L. 1984. The humanT cell receptor. Ann. Rev. Irnmunol. 2:23-50 75. Krangel, M. S. 1987. Endocytosisand recyclingof the T3-Tcell receptor complex. Therole of T3 phosphorylation. J. Exp. Med. 165:1141-59 76. Minami,Y., Sanmlson,L. E., Klausner, R. D. 1987.Internalization andcycling of the T cell antigen receptor: role of protein kinase C. J. Biol. Chem.262: 13342-47 77. Adkins,B., Mueller,C., Okada,C. Y., Richert, R. A., Weissman, I. L., Sprangmole, G. J. 1987.Early eventsin T cell maturation. Ann. Rev. Immunol. 5:325~55 78. Mathieson, B., Sharrow, S., Rosemberg, Y., Hammerling, U. 1981. Lyl+23- cells appear in the thy+ cells. Nature289: musbefore Lyt123 179

79. Kingston, R., Jenkinson, E. J., Owen, J. J. T. 1985. A single stem cell can recolonize an embryonicthymus,producing phenotypicallydistinct T cell populations. Nature317:811 80. Fowlkes,B. J., Edison, L., Mathieson, B. J., Chused,T. M.1985.Early T lymphocytes:differentiationin vivoof adult intrathymic precursor cells. J. Exp. Med. 162:802-22 81. Haars, R., Kronenberg,M., Gallatin, W.M., Weissman,I. L., Owen,F. L., Hood, L. 1986. Rearrangement and expression of T cell antigen receptor and ~, genes during thymic development. J. Exp. Med.164:1 82. Samelson, L. E., Londsten, T., Foulkes,B. J., van denElsen, P., Terhorst, C., Druis, M. M., Germain,R. N., Schwartz,R. M. 1985. Expression of genesof the T-cell antigen receptor complexin precursor thymocytes.Nature 315:765~58 83. van Dongen,J. J. M., Quertermous,T., Bartram,C. R., Gold, D. P., WolversTettero, 1. L. M., Comans-Bitter,W. M., Hooijkaas,H., Adriaansen,H. J., de Klein, A., Raghavachar,A., Ganser, A, Duby, A. D., Seidman,J. G., van denElsen, P., Terhorst,C. 1987.T cell receptor-CD3complexduring early T cell differentiation: analysis of immature T cell acute lymphoblasticleukemias (T-ALL) at DNA,RNA,and cell membrane level. J. Immunol.138: 1260-69 84. Furley, A. J., Mizutani,S., Weilbaeeher, K., Dhaliwal,H. S., Ford, A. M., Chan, L. C., Molgaard,H. V., Toyonaga, B., Mak,T., van den Elsen, P., Gold, D., Terhorst, C., Greaves,M.F. 1986. Developmentally regulated rearrangementand expression of genes encoding the T cell receptor-T3 complex. Cell46:75-87 85. van den Elsen, P., Burns,G., Gerhard, D. S., Pravtcheva,D., Jones, C., Housman, D., Ruddle, F., Orkin, S., Terhorst, C. 1985. Assignmentof the genecodingfor the T3-6subunit of the T3-T-cell receptor complexto the long arm of humanchromosomei 1 and to mouse chromosome 9. Proc. Natl. Acad. Sci. USA82:2920-24 86. Gold, D., van Dongen,J., Morton,C., Bruns, G., van den Elsen, P., Van Kessel, A., Terhorst,C. 1987.Thegene encodingthe ~-subunitof the T3/T-cell receptor complexmapsto chromosome 11 in humansand to chromosome 9 in mice. Proc. Natl. ~1cad. Sci. USA84: 1664~8 87. Tunnacliffe,A., Buluwela,L., Rabbitts,

Annual Reviews

Annu. Rev. Immunol. 1988.6:629-662. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

THE TCR/CD3 COMPLEX661 T. H. 1987. Physical linkage of three CD3 genes on human chromosome 11. EMBO J. 6:2953-57 88. Saito, H., Koyama,T., Georgopoulos, K., Clevers, H., Haser, W.G., LeBien, T., Tonegawa,S., Terhorst, C. 1987. Close linkage of the mouseand human CD3-7and ~ genes suggests a control of their transcription by common regulatory elements.Proc.Natl. Acad.Sci. USA.In press 89. van den Elsen, P., Georgeopoulos,K., Shepley,B. A., Orkin,S., Terhorst,C. 1986. Exon/intronorganization of the genes coding of the 6-chains of the humanand murine T cell receptor/T3 complex. Proc. Natl. Acad. Sci. USA 83:2944-48 90. Kabat,E. A., Wu,T. T., Reid-Miller, M., Perry, H. M., Gottesman, K. S. 1987. Sequencesof Proteins of Immunological Interest. Washington,DC: USDep. Health Hum.Serv. 91. van Dongen,J. J. M., Krissansen, G. W., Wolvers-Tettero, I. L. M., Comans-Bitter, W., Adriaansen, H. J., Hooijkaas, van Wering, E. R., Terhorst, C. 1987. Cytoplasmic expression of the CD3antigen as a diagnostic markerfor immatureT cell malignancies.Blood.In press 92. Meuer,S. C., Hussey,R. E., Fabbi, M., Fox, D., Acuto,O., Fitzgerald, K. A., Hodgdon,J. C., Protentis, J. P., Schlossman, S. F., Reinherz, E. L. 1984. Analternative pathwayof T-cell activation: Afunctionalrole for the 50 kd T11sheep erythrocytereceptor protein. Cell 36:897-906 93. Siliciano, R. F., Pratt, J. C., Schmidt, R. E., Ritz, J., Reiiaherz,E. L. 1985. Activation of cytolytic T lymphocyte andnatural killer cell functionthrough the T11sheep erythrocytebinding protein. Nature428-30 94. Pantaleo, G., Olive, D., Poggi, A., Kozumbo, W.J., Moretta, L., Moretta, A. 1987. Transmembrane signalling via the T11-dependentpathway of human T cell activation. Evidencefor the involvementof 1,2-diacylglycerol and inositol phosphates.Eur. J. Immunol. 17:55~50 95. Fox, D. A., Hussey,R. E., Fitzgerald, K. A., Bensussan, A., Daley, J. F., Schlossman, S. F., Reinherz, E. L. 1985. Activation of humanthymocytes via the 50 kd Tll sheep erythrocyte binding protein induces the expression + of interleukin 2 receptors on both T3 and T3- populations. J. Immunol.134: 330-35 96. Ramadi,D., Fox, D. A., Milanese,C.,

Reinherz,E. L. 1986. Selective inhibition of interleukin 2 genefunction following thymocyte antigen/major histocompatibility complexreceptor crosslinking: Possiblethymicselection mechanism.Proc. Natl. Acad.Sci. USA 83:7008-12 97. Alcover, A., Ramarli, D., Richardson, N. E., Chang,H. S., Reinherz, E. L. 1987. Functional and molecular aspects of humanT-lymphocyteactivation via T3/Ti and Tll pathways. lmmunol.Rev. 95:5-37 98. Dembic,Z., Haas, W., Zamoyaska,R., Parnes, J., von Boehmer,I. T., Steinmetz, M.1987. Transfectionof the CD8 gene enhances T cell recognition. Nature 326:510-11 99. Gabert,J., Langlet, C., Zamoyska, R., Parnes, J. R., Schmitt-Verhulst,A. M., Malissen, B. 1987. Reconstitution of MHC-class I specificity by T cell receptor and Lyt-2 genetransfer. Cell 50.: 545-54 100. Sleckman,B. P., Peterson, A., Jones, W.K., Foran, J. A., Greenstein,J. L., Seed, B., Burakoff, S. J. 1987. Expression and function of CD4in a murineT cell hybridoma.Nature328: 351-53 101. Gay,D., Maddon, P., Sekaly,R., Talle, M. A., Godfrey, M., Long, E., Goldstein, G., Chess, L., Axel, R., Kappler, J., Marrack,P. 1987. Functional interactions betweenhumanT cell protein CD4and the majorhistocompatibility complexHLA-DR antigen. Nature 328:626-29 102. Hansen,J. A., Martin,P. J., Nowinski, R. C. 1980. Monoclonal antibodies identifying a novelT cell antigen and Ig antigens of humanlymphocytes. lmmunogenetics10:225-29 103. Ledbetter, J. A., June, C. H., Grosmarie, L. S., Rabinovitch,P. S. 1987. Crosslinkingof surfaceantigens causes mobilization of intracellular ionized calciumin T lymphocytes.Proc. Natl. Acad. Sei. USA84:1384-88 104. Moretta,A., Poggi,A., Olive, D., Bottino, C., Fortis, C., Pantaleo, G., Moretta, L. 1987. Selection and characterization of T-cell variants lacking moleculesinvolvedin T-cell activation (T3 T-cell receptor. T44, and Ttl): analysis of the functional relationship amongdifferent pathwaysof activation. Proc. Natl. Acad.Sci. USA84: 1654-58 105. Kroczek, R. A., Gunter, K. C., Germain,R. N., Shevach,E. M. 1986. Thy-1 functions as a signal transduction moleculein T lymphocytesand

Annual Reviews

662

CLEVERSET AL

Annu. Rev. Immunol. 1988.6:629-662. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

transfected B lymphocytes.Nature322: pathwaysin a T c~ll receptor alpha 181-84 chain deletion variant of a cytolytic T c¢11 clone. Nature325:628-31 106. Gunter, K. C., Germain, R. N., Kroczek,R. A., Saito, T., Yokoyama, 108. Nguyen,C., Mattei, M.-G., Mattei, W.M., Chan, C., Weiss, A., Shevach, J.-F., Santoni, M.-J., Goridis, C., E. 1987. Thy-1mediatedT cell actiJordan,B. R. 1986.Localizationof the vation requires coexpressionof CD3/Ti human NCAM gene to band q23 of complex. Nature 326:505-07 chromosome 11: the third gene coding for a cell interaction moleculemapped 107. Schmitt-Verhulst, A. M., Guimezaes, to the distal portionof the long armof A., Boyer, C., Poenie, M., Tsien, R., Buferne, M., Hua, C., Leserman, L. chromosome 11. J. CellBiol. 102: 71115 1987. Pleiotropic loss of activation

Annu. Rev. Immunol. 1988.6:629-662. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

Annu. Rev. Immunol. 1988.6:629-662. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

Annual Reviews www.annualreviews.org/aronline

Annu. Rev. Immunol. 1988.6:663-688. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

Ann. Rev. Immunol.1988.6: 663~8

THE T CELL RESPONSE TO THE MALARIA CIRCUMSPOROZOITE PROTEIN: An Immunological Approach Ito Vaccine Development Michael F. Good,’~ Jay A. Berzofsky,* Louis H. Miller~f

and

~" TheLaboratoryof Parasitic Diseases, NationalInstitute of Allergyand Infectious Diseases, NationalInstitutes of Health, Bethesda, Maryland 20892 * TheMetabolismBranch, National CancerInstitute, National Institutes of Health, Bethesda, Maryland20892 Introduction Aneffective malarial vaccine wouldsave the lives of over one million children annually and reduce the suffering of hundreds of millions of children and adults in tropical countries throughout the world (1). present, there is no vaccineagainst any humanparasitic disease, yet vaccines against other pathogens have had an enormousimpact on public health. Whilelaboratory animals can be successfully vaccinated against malariausing crude parasite antigens (reviewedin 2), such an approach not feasible for humans becauseof the impracticality of large-scale culture (3) and the cost and associated risks of using blood- or serum-derived products in a humanvaccine. Unless such problems can be overcome, malaria vaccines suitable for humanuse will have to be producedusing recombinant DNA or synthetic peptide technology. Indeed, candidate recombinant (4, 5) andsynthetic (6, 7) vaccineshavealreadybeentested humansand nonhuman primates; although results have been encouraging, these humanvaccines (5, 7) to date have had a limited efficacy. These ~ The USGovernmenthas the tight to retain a nonexclusive, royalty-free license in and to any copyright coveting this paper.

663

Annual Reviews

Annu. Rev. Immunol. 1988.6:663-688. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

664

GOOD, BERZOFSKY & MILLER

vaccines, which contain a limited amount of parasite information, are referred to as subunit vaccines. In this review, we trace the development of a subunit vaccine for the sporozoite stage of the life cycle of the malarial parasite. Becausemalaria immunityis stage-specific, only sporozoite-specific antigens can Joe considered for a sporozoite vaccine. Althoughsignificant progress towards a sporozoite vaccine has been made, we nowrealize that the parasite has developed clever ways to evade immunecontrol. The lessons we are learning from the sporozoite vaccine saga should prove valuable for the rational developmentof vaccines aimed at other stages in the life cycle, and indeed at other parasites. Malaria is initiated with the bite of an infectious mosquito. Sporozoites (hundreds to a few thousand) are inoculated into the subcutaneous tissue or directly into a capillary during a blood meal (Figure 1). Sporozoites travel through the circulation, from which most are cleared in a few minutes by the spleen or liver. Theyenter hepatocytes in the liver, probably via a specific receptor (8, 9), and develop by schizogony over a number days. Sporozoites also interact with Kupffer cells in the liver (10). For Plasmodiumfalciparum(the most deadly of the humanmalarias), hepatic schizogony lasts approximately one week, after which thousands of merozoites are liberated to invade red blood cells. The red blood cell stage is responsible for all the clinical manifestations of malaria. Eventually, sexual stage parasites (gametocytes) develop in the red blood cells. These are taken up by mosquitoesduring a blood meal and fertilize and develop in the midgut. Immature sporozoites develop in a couple of weeks and migrate through the hemolymph to the mosquito salivary glands where they mature. During this maturation, sporozoites develop a coat protein, the circumsporozoite (CS) protein. It has been knownsince 1941 (1 l) that immunization with sporozoites could lead to stage-specific protection from malaria, i.e. protection against sporozoite challenge but not against blood-stage challenge. Morerecently, these findings were extended to include a numberof different host-parasite systems, and more detailed immunological analyses were performed (reviewed in 12 and 13). Manyof the special requirements for effective sporozoite immunization are not understood. For example, effective immunization of adult mice requires intravenous inoculation with irradiated but not heat-killed sporozoites; this is consistent with the hypothesis that the sporozoites mayhave to travel to and then penetrate hepatocytes to induce immunity.Non-irradiated sporozoites are also effective, but the animals have to be given chemoprophylaxis following immunization to kill the asexual erythrocytic parasites. Other routes of immunizationare ineffective, except in juvenile mice, where intramuscular administration of

Annual Reviews

Annu. Rev. Immunol. 1988.6:663-688. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

MALARIA

VACCINE

DEVELOPMENT

665

IN MOSQUITO GUT

GAMETOCYTES

Figure 1 The life

cycle of malaria.

sporozoitesis effective. Immaturesporozoites, taken fromthe oocysts in mosquitomidguts, are not effective immunogens. As mentioned,immature sporozoites lack the CSprotein coat but probablyhavea numberof other antigenic differences as well, whichmayexplain their lack of immunogenicity. Furthermore,immaturesporozoitesare not infective, and antigensexpressedfollowinginfection of liver cells maybe requiredfor induction of immunity.Followingeffective immunization,sporozoite immunity, whichis species-specific, wanesafter about three months. The CS Protein TheCSprotein wasfirst identified as a candidateantigen for protective immunityin 1980. Immuneserum immunoprecipitated a 44-kd antigen

Annual Reviews

Annu. Rev. Immunol. 1988.6:663-688. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

666

GOOD, BERZOFSKY & MILLER

(since shown to the CS protein) from the surface of sporozoites of beryhei 04). A monoclonal antibody, which recognized the same antigen, was then shownto passively transfer protection to naive mice (15). The CS protein (Figure 2) is the major sporozoite protein and covers the sporozoite surface. Genes for CSproteins have been cloned for simian (16-18), routine (19-22), and humanmalarias (8, 23-28). The sequence for a P.falciparum CS protein (7G8 clone) was published in 1984 by Dame et al (8). The protein contains 412 amino acids and has a structure which resembles CS proteins from other species of malaria. The central third of this and other CS proteins consists of multiple repeating sequences. The major repeating sequence for P. falciparum is Asn-Ala-Asn-Pro. This is repeated approximately 40 times. A minor repeating sequence, Asn-ValAsp-Pro, occurs a few times in P.falciparum. The repeating sequences are unique for each species. For example, for P. vivax, the repeating sequence is Gly-Asp-Arg-Ala-Asp-Gly-Gln-Pro-Ala.The central repetitive segment is the immunodominant B cell epitope of the CS protein and, indeed, of the sporozoite. Antibodies directed against this repetitive epitope can block sporozoites from entering cultured hepatocytes (29-31) and, for murine malaria, can passively protect animals from sporozoite challenge (15, 32). While the repetitive epitope varies amongmalarial species, it appears to be conserved within species, with the exception of P. knowlesi (17) and cynomolyi(18), simian malarias. Thus, for manydifferent isolates (including six clones) of P. falciparum (33, 34), the immunodominant repeat (NANP)n.It was this markedconservation and the fact that anti-(NANP)n antibodies blocked sporozoite invasion of hepatocytes in vitro (29-31) that madethe repetitive epitope an attractive vaccine candidate. The regions which flank the repeats contain two smaller segments that are strongly conserved amongdifferent species of malaria. These two regions are referred to as Region I (immediately amino terminal to the repeats) and Region II (located approximately 60 amino acids carboxyl terminal to the repeats). It has been suggested that these regions are conserved for important functions of the protein such as receptor binding for cell invasion (8). Antibodies against a Region I peptide.blocked sporozoite invasion of liver cells in vitro (9). Curiously, Region II bears Signal I

Repeats RI B kwwvv~Arvv~

R]I

Anchor

m

I

P. falciparum:majorrepeatNANP minorrepeat NVDP Figure 2 Diagrammaticrepresentation of the CSprotein. RI and RII refer to Region I and Region II, regions conserved between different species.

Annual Reviews

Annu. Rev. Immunol. 1988.6:663-688. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

MALARIA VACCINE

DEVELOPMENT

667

striking homology to the adhesiveprotein thrombospondin (35), indicating that RegionII maybe involved in cell adhesion. Suchhost homologies mayalso be one wayto reduce the antiparasite immune response. Since the sequenceof a cloned P. falciparurn CSprotein wasdetermined (8), the sequences of three other isolates of/~. falciparum have been determined(26, 28). Thesequenceswereall remarkablyconserved,differing in only 9 aminoacid positions from the morethan 230 outside of the repetitive region; however,all mutationsat the nucleotidelevel resulted in codingchanges, leading de la Cruzet al (28) to suggest that pressure the protein level, possibly immunepressure, was responsible for these mutations(see below).

Mechanismsof Sporozoite Immunity In 1980, it was shownthat a monoclonalantibody against the CS protein could transfer protection in a murinemodel(15). Subsequently,it was shownthat antibodies raised against the major repetitive epitope, (NANP)n, of P.falcipariumcould block entry of sporozoites into a cultured hepatomacell line (29-31). Thus,an early rationale for vaccine development was to develop an immunogenthat could induce anti-(NANP)n antibodiesin vivo. Antibodiesto the repetitive epitope of the sporozoiteantigen following immunizationmaygive only limited protection (32) although monoclonal ~/ntibodiesagainst the CSprotein repetitive epitopecan transfer protection [10/~g being required in one study (15), and 100 #g of a different monoclonal antibody required in another study (32)]. Neither immuneserum (36) nor immune B cells (32) can transfer this protection. Furthermore, a study from East Africa (37), whereadults were given malaria chemotherapyand their bloodparasitemialevels werefollowedduring the transmissionseason,susceptibility to malariadid not correlate with the level of antisporozoite antibodies. Thus, one approach to vaccine development is to develop an immunogen capable of eliciting a very high antibody responge--a response higher than that normallyelicited by sporozoites duringnatural infections. First stage vaccinetrials in humans(5, 7) have shownthat this approach has somemerit, although success has been reported in only two individuals from nine challenged with viable sporozoites. It is worthnotingthat in a mousevaccinetrial (32), immunization with a P. berghei CS peptide afforded limited protection whenthe mice were challenged with 500 sporozoites, but no protection whenthey were challengedwith 1000or 5000sporozoites. Thesemicehad high titer antiCSprotein antibodies. Conversely,mice immunizedwith irradiated sporozoites wereprotected fromchallenge with 5000sporozoites. Anotherapproach to sporozoite vaccine developmentis to develop an

Annual Reviews

Annu. Rev. Immunol. 1988.6:663-688. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

668

GOOD, BERZOFSKY & MILLER

immunogen that moreclosely mimicsimmunizationwith irradiated sporozoites. Animportant consideration is to prime a population of immuneT cells. Thefinding that athymicmice cannot be protected by immunization with sporozoitesis a strong indication that T cells are requiredfor immunity (38, 39). Conversely,the finding that #-suppressedmice, whichlack both B cells and circulating immunoglobulin,can be successfully immunized with sporozoites demonstratesthat effector T cells are sufficient for natural sporozoite immunity(38). This result was confirmedby the demonstration that T cells could transfer immunityfrom sporozoiteimmunized animals (32, 40). Furthermore,as was observedby Spitalny al workingin the murinesystem (41) and Clyde et al workingwith the humanparasite, P. falciparum (42), sporozoite immunityprecedes the developmentof circumsporozoite precipitin antibodies. The present difficulty with developingvaccinesto primeT cells for sporozoiteimmunity is the lack of definition of the protective antigenof T cell immunity. The enormousinterest in the CSprotein as a sporozoite vaccine candidate comesfrom its identification as the major B cell antigen of the sporozoite and the target of neutralizing antibodies (14, 15, 29-31). covers the surface of the sporozoite (43) and binds most antisporozoite antibodies (44). The CSprotein is the only sporozoite protein for which the genehas beencloned, but whetherit stimulates protective T cells is unknown.At present the only way to address this issue is with circumstantialevidenceas presentedin sections below. Effector T cells must workby either a cellular cytotoxic mechanism (CD8cells predominantly, but also CD4cells), or by elaboration of a parasiticidal lymphokine [e.g. 7-interferon (IFN), whichhas been showncapable of eradicating liver stage schizonts (45, 46)]. It is importantthat T cells will not recognize sporozoites but will only recognizeprocessed sporozoite antigens, presented in association with MHC antigens. Thus, CD4T cells could recognize sporozoite T epitopes in association with class II major histocompatibility complex(MHC)antigens on the surface of Kupffer cells (liver macrophages).CD4T cells could secrete ?IFNto act in the local environmentand possibly kill a sporozoite-infected hepatocyte. Alternatively, a CD8T cell could recognizeT epitopes in association with class I MHC antigens on the surface of an hepatocyte and could directly kill that hepatocyte. Evidencesupporting the possibility that the CSprotein is an immunogen of such CD8effector T cells is the observation that the CSprotein is located inside infected hepatocytes(47). In other systems, e.g. influenza (48), the foreign proteins expressedinside the host cells are processed to becomethe natural immunogens for class 1-restricted cytotoxicT cells. Indeed,recently a role for cytotoxicT lymphocytes (CTL) in sporozoite immunityhas been defined, and it is likely that infected

Annual Reviews

Annu. Rev. Immunol. 1988.6:663-688. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

MALARIA VACCINEDEVELOPMENT 669 hepatocytes which bear class I antigens are targets for CTL(49). Weiss al demonstratedthat in vivo depletion of CD8(class I-restricted) T cells, but not CD4(class II-restricted) T cells, abrogated sporozoite immunity in a murine model(Table 1). Similar findings were independently reported by Schofield et al (50). Developmentof a CD8T cell immuneresponse would be helped by the participation of CD4helper T cells which maybe primed by CS-specific T cell epitopes and other sporozoite T cell epitopes following processing by Kupffer and other Ia-bearing cells; however, it is knownthat CD8immunity to ectromelia virus can develop in vivo in the absence of CD4cells (51). T Cell Epitopes

of the CS Protein

T cell epitopes can be divided into those recognized by CD4T cells and those recognized by CD8T cells. While the peptide epitopes recognized by these two distinct T cell populations are processed differently by antigen presenting cells (48), the epitopes themselves probably have similar physicochemicalproperties (52-54) and are recognized by T cells in similar ways. Wehave initially examined CD4epitopes on the CS protein. CD4 T cells are primarily helper cells for B cells or CD8cells but are also effector cells in that they can secrete parasiticidal lymphokines(e.g. ?IFN) and mediate specific cytotoxicity (55, 56). Since most CD4T cells proliferate following activation, a simple assay for CD4activity is antigeninduced proliferation. Another commonassay is B cell help, i.e. antibody production. While clonal heterogeneity exists with respect to CD4T cell function (57-59), it is probably reasonable to assume that, at the popu-

Table1 Theeffect of anti-T cell treatmentsonsporozoiteimmunity Daysto detectable parasitemia aImmune status Normal Immune Immune Immune Immune Immune

In vivotreatment

Infected/Total

Median Range

none none anti-CD8(mouseIgG2a) control mouseIgG2a anti-CD4(rat IgG2b) controlrat serumIgG

15/15 0/10 9/9 0/5 0/5 0/5

4-7 5 (not detected) 5 5-6 (not detected) (not detected) (not detected)

aMice received4 dosesof irradiatedsporozoites (immune), or noimmunization (normal) prior treatment, andbloodsmears weretakenbeginning onthethirddayafter challenge with5000viable sporozoites. Summarized fromWeiss et al (49)withpermission.

Annual Reviews

670

GOOD, BERZOFSKY & MILLER

Annu. Rev. Immunol. 1988.6:663-688. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

lation level, help for B cells and CD8CTLis induced concomitantlyby the samestimulus. THEMURINE T-CELLRESPONSE One humansporozoite vaccine candidate was R32tet~, a fusion protein consisting of 32 repeats [(NANP)~sNVDP]2 from the CS protein of P.falciparumfused to a peptide correspondingto part of a tetracycline resistance generead out of frame (29). Toassess its suitability as a vaccine, wesearchedfor T cell epitopes on this molecule(60). Different H-2congenicmiceon the B 10 background were immunized with R32tet32or with (NANP)6 (Table 2). Of all strains immunized with (NANP)6, only mice bearing the I-Ab gene could generate anti-(NANP)nantibodies. Similarly, only lymphnode cells from immunized I-Ab-bearingmice could proliferate to (NANP)n in vitro. Del Giudice et al (61) and Tognaet al (62) had similar findings, using (NANP)4o an immunogen.Wehave since examinedfurther B10 congenic strains, expressing amongthem 14 different class II genes, and have found that only I-Ab bearing strains can respondto (NANP)n. Wethen looked at the bresponse from mice immunizedwith R32tet32 and also found that/-A bearing strains could respond to this larger immunogen. Other strains [B10.D2(I-Ad, I-Ed), BI0.HTT(I-As, I-E~kE~)] also responded. These strains wereclearly respondingto the tet32 segmentof R32tet32or a T cell site formed by the junction of NVDP and tet32; however, a numberof strains (B10.BR,B10.S, B10.GD)did not respond even to this larger Table 2 Immuneresponse to a first a(Units/ml)]

stage sporozoite vaccine (R32tet32) [Anti-NANPIgG

Immunogen

H-2 alleles Strain

K

A

B

J

E

C

S D (NANP)6

B10.BR B10.A(4R) B10.HTT B10.S

k k s s b b d d

k k s s b b d d k

k b s s b b d b

k b s s k b d b k

k (b) k (s) k (b) d (b) k

k b k s d b d b

k b k s d b d b

k

k

B10.A(5R) B10 B10.D2 B10.GD B10.MBR

b

k

k b d s d b d b q

R32tet32

<1
<1 ND

<1 <1 67 73

370 <1 274 587

<1 ND <1

80 10 ND

a Mice were immunizedintraperitoneally with 80 #g of immunogen emulsified in CFAon day 0 and boosted with 10/~g of aqueousimmunogen on days 21 and 31. Sera from a day 41 bleed were used, and specific IgGlevels compared with a control serumdefinedto contain 1000units of specific IgGper ml, using an ELISAassay. Data summarized from Goodet al (60) with permission.

Annual Reviews

Annu. Rev. Immunol. 1988.6:663-688. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

MALARIA VACCINE

DEVELOPMENT

671

immunogen.Wesuggested that if a similar situation occurred in humans, few would respond well to this vaccine candidate, R32tet32, because of genetic restriction. Morerecently, the results of the first humanvaccine trial using R32tet32 were published. Of 15 individuals inoculated, only one developed significant anti-(NANP)ntiters (5). While it is tempting speculate that this low human immuneresponsiveness was due to human immune response (lr) gene control, this could not be proven without detailed family studies involving HLAgenotyping. Other factors--for example, poor adjuvanticity--could also account for this low response. On the basis of our initial murine studies, we reasoned that it was necessary to examinethe flanking, nonrepetitive, regions of the CSprotein for the presence of T cell antigenic sites. Wefirst determinedwhich mouse strains were high antibody responders to the CS protein as encoded by a recombinant vaccinia virus. To our surprise, only mice bearing I-Ab or I-Ak MHC alleles were high responders (63, 64). Manyother strains, each of which expresses two functional class II Molecules, were nonresponders or low responders. As a control, all strains used in the experimentproduced equivalent titers of vaccinia-specific antibodies. In these experiments, the recombinant virus was used because there was no source of purified CS protein, and sporozoites of the 7G8 strain [whose CS protein has been sequenced(8)] were very difficult to produce in the laboratory. Wewere not surprised that I-Ab-bearing mice responded well to the CS protein, since the T cells of these mice had been shownto respond to the (NANP)nepitope that comprises approximately one third of the protein. However, I-Ak-bearing mice [B10.BR, B10.A(4R)], which do not respond to (NANP)n(Table 2), responded just as well to the CS protein. To locate the potential antigenic site recognized by I-Ak-bearing mice, we madeuse of the observation that most T cell epitopes can be defined by linear peptide sequences which have the potential to fold as amphipathic alpha helices (65-67), i.e. helices which present a hydrophobicface as well as a hydrophilic face. Using a computerprogram(67) to locate such potential T cell sites on the CS protein, we observed that a major predicted site occurred immediately amino terminal to Region II. This predicted site had the highest amphipathic score for the entire molecule (64). Wewere curious to know whether the l-Ak-bearing mice had T cells that were responding to the predicted T cell site. Undernormalconditions, this question could be answered by immunizingmice with the native protein and then challenging draining lymph node cells in vitro with a peptide corresponding to the putative T cell site. However, we had no source of purified CS protein, and vaccinia virus does not function as an immunogen in this assay. Thus, to answer this question, we took advantage of the fact than an anamnestic response occurs in animals whose T cells have been primed. Thus, B10.BR

Annual Reviews 672

GOOD, BERZOFSKY & MILLER

Annu. Rev. Immunol. 1988.6:663-688. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

(H-2 k)

mice were immunizedwith a synthetic peptide corresponding to this putative T cell site, or adjuvantalone, and6 weekslater the micewere infected with the CS-vacciniavirus. If T cells had been primedby the peptide, and if those T cells recognizedthe T cell site in the recombinant CS protein, a secondary anti-(NANP)nantibody response should occur after CS-vacciniainoculation. This indeed did occur (64), and further k] studies showedthat this peptide also primedT cells in B 10.A(4R)[I-A mice,whichdo not possessan I-E molecule,indicating that this T cell site wasI-Ak-restricted (Figure 3). This peptide, whichwereferred to as Th2R because it was the second region of the CS protein shownto stimulate Thcells, also stimulatedproliferating T cells in I-Ak-bearingstrains. We confirmedthat Th2Rwas indeed a helper T cell site by constructing a synthetic immunogen, Th2R-NP(NANP)sNA, and demonstrating that this, but not NP(NANP)sNA alone, was capable of inducing high titer anti-(NANP)nantibodies in B10.BRand B10.A(4R)mice (64). thus successfullypredicted a majorT cell site on the CSprotein and used it to construct a synthetic immunogen containing defined T and B cell epitopes from the sameprotein. AlthoughI-Ab and/-Ak-bearingmice were the high responders to the CSprotein, and although we had located I-Ab and I-Ak-restricted helper Tcell sites on the CSprotein, there mightbe other similarly restricted sites elsewhereon the protein. To address this, Pomboet al (68), tolerized b) and B10.BR(H-2 k) mice to the respective T cell sites neonatal B10(H-2 and then immunizedtolerant mice with the CS protein. To our surprise, tolerant micewere able to respondto the CSprotein, an indication that other helper T cell sites werelocated on the CSprotein. Tolocate all of these sites, weconstructedsynthetic overlappingpeptides spanningthe entire CSprotein (Figure 4). Initially, B10and B10.BR mice were immunizedwith groups of peptides and subsequently infected with the CS-recombinant vaccinia virus. Eight days post infection, serumanti(NANP)n antibody titers were measuredand comparedwith titers from mice injected with adjuvant alone and subsequently infected with the virus. For both strains, peptides fromthe carboxylterminal region of the moleculewere T cell antigenic sites. The experimentwasthen repeated with individual peptides to locate the sites precisely. For B10.BR mice, a major site apart from Th2Rwas located, and for B10mice, a major site apart from (NANP)~ waslocated (69). Both of these sites are capable folding as amphipathichelices. THEHUMAN T-CELL RESPONSE Whilewe had determined major T cell sites recognized by mice, wecould only speculate as to whether humanswould see these sameT cell sites. Theliterature is very limited as to whetherT

Annual Reviews MALARIA VACCINE DEVELOPMENT

I

I

I

I

"I~

673

~

Annu. Rev. Immunol. 1988.6:663-688. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

1.0

B10.D2

Log2~lufion of ~um Figure 3 B10.BR, B10, B10.D2, and B10.AOR ) mice (five per group) were immunized with peptides (100 #g) emulsified in CFA, and 6 weeks later they were challenged with the CSvaccinia virus by scarification. Nine days after challenge, sera were collected and antibodies to (NANP)nwere measured in the pooled sera from each group. The peptides used for the immunization were as follows: IB, Th2R [PSDKHIEQYLKKIKNSIS(C)],representing amino acids 326 to 343 from the sequence of the CS protein from the 7G8 strain of P. falciparum, plus an extra cysteine at the carboxyl terminus; ~,, EKLRKPKHKKLKQP, representing amino acids 103 to 116; O, KPKHKKLKQPGDGNPDPN, representing amino acids I07 to 124; O, saline. The B10.A(4R)mice were tested only with Th2Rto mapthe genetic regulation of the positive response in B10.BRmice. Reproduced from Goodet al (64) with permission.

cells from humans and mice recognize the same immunodominantsites; there is only one example of similar recognition, for influenza hemagglutinin (70, 71). Thus, in collaboration with scientists from the Medical Research Council Laboratories in The Gambia, we tested the in vitro reactivity to the overlapping synthetic peptides spanning the entire molecule, of peripheral blood lymphocytes from adult Gambiansliving in a region holoendemic for Plasrnodium falciparurn (72). These individuals

Annual Reviews 674

GOOD, BERZOFSKY & MILLER

7 6 4

5

3

MMRK LA I

10 Signal Sequence20 30 L SVSSFL FVEAL FQEYQCYGSSSNTRVL

40 NE LNYDNAGTNL

GO YNEL 13

60 EMNYYGKQE

10 9

Annu. Rev. Immunol. 1988.6:663-688. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

NW¥ S L KKNSf~

SL GE NDDGNNNNGDNGREGKDEOKRDGNNE DNEK L RKPKHKK

LKQPGDGN (A) (D)

17 (NPNA)~ L:x30 300 NPNKNNOGNGQGHNMPNDPNRNVDE 24 ~3 22

CENTRALREPEATS

320 33O 340 NANANNAVKNNNNEEPSDKH t EQY,L K K t KNS I (G~ (O) (K) tO) 29

25 DELDYEND I

(o)

~0 EKK I

350 STEWSPCSVTCGNG I

36O QVR I

310 ~

370 KPGSANKPK (D)

26 ~90 CKMEKCSSVFNVVNSS I

400AnchorSeque~ce 410 GL I MVL SF L F LN

Figure4 Schematicrepresentation of the 29 peptides studied, using single letter aminoacid code (64). Each peptide consisted of 20 aminoacids from the 7G8clone of the CS protein (8), as shown,plus a carboxyl terminal cysteine. (NPNA)6is peptide 14. Letters under the mainsequence identify variant residues (26, 28). FromGoodet al (72), with permission.

wouldhavebeenexposedto numerous inoculations of sporozoitcs during their life time. Toour surprise, a significant proportionof individuals (approximately40%)did not respondto any of the synthetic peptides; however,in those whodid, the majorresponseoccurredto peptideslocated carboxylterminalto the central repeats, as wehadfoundin mice. Thetwo peptides giving the greatest frequencyof responsewerepeptides 361 to 380, and326 to 345 (Th2R).Bothof these are predictedby the helical amphipathicity algorithm.Curiously,the responseto a RegionII peptide (located in the carboxylterminalregion of the molecule)wasminimal, althoughthe responseto peptides immediatelyaminoterminalandcarboxylterminalto RegionI! washigh. RegionII is known to be extensively homologous to a platelet-derivedfactor, thrombospondin (35); it is possible that host mimicry andresultantT cell toleranceis a parasiteimmune

Annual Reviews MALARIAVACCINEDEVELOPMENT

675

Annu. Rev. Immunol. 1988.6:663-688. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

evasion mechanism.Weobserved that not all adult individuals in this holoendemicarea had serum antibodies that could bind (NANP)n. fact, only about 40%of Gambianshad serumtiters of 2048 or greater. Furthermore, we were unable to find any correlation between antibody titer andthe T cell responseto anygiven peptide. Of greater significance, a numberof people whohad serumantibodies did not respond to any of the peptides whosesequenceswere based on the 7G8clone. This strongly suggestedthat T cell help in those individuals was providedby response to a T epitope froma variant CSprotein sequence(see below). PARASITE POLYMORPHISM A closer inspection of sequences from three other CS proteins (26, 28) revealed that the immunodominant sites for humanT cell recognition correspondedprecisely to the polymorphicsegmentsof the molecule(72). As noted by de la Cruzet al (28), there nine aminoacid variations amongthe sequencedCS proteins, and there are no silent mutationsat the nucleotide level in the flanking regions of the molecule;these facts stronglysuggestthat pressureat the protein level dictates the variation. Ourfindings also (Figure 5) strongly suggestedthat this pressure camefromimmune T cells; this confirmedprevioussuspicions (28). An inspection of the sequencevariation amongthe published CSprotein sequences(Figure 4) reveals that the variation is centered around 15-

10-

5-

1 2 3 4 5 6 7 8 9 1011121314151617181920212223242526272829 PEPTIDE NUMBER Figure 5 Histogram indicating the number of people from 35 tested whoresponded to each individual peptide. Shaded bars are those in which mutations from the 7G8sequence have been observed [Refs. 26, 28; peptide 26 contains an unpublished variant residue (V. F. de la Cruz, A. Lal, T. F. McCutchan)]. The frequency of response to peptides associated with mutation was compared to the frequency of response to the other peptides. The median number of responses to mutation-associated peptides was 5.5 compared to a median of two responses to the other peptides. The difference betweenthe respective medians is significant (Wilcoxon test, U = 48.5, p = 0.016). FromGoodet al (72), with permission.

Annual Reviews

Annu. Rev. Immunol. 1988.6:663-688. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

676

GOOD, BERZOFSKY & MILLER

peptides 13, 17, 20, and 24. Antibodiesagainst the CS-protein in the region ofpeptide 13 can block sporozoite invasion (9), and variation in this region may represent a mechanism of escape from antibody-mediated immunity (28); however, peptides 17, 20, and 24 are humanimmunodominant T sites (Figure 5). Because of overlap, manyof the peptides from the carboxyl terminal segment of the molecule represent variant regions. Indeed, when the frequency of response to peptides associated with variation was compared with the frequency of response to other peptides, the difference was significant (p = 0.016). Our studies had been performed with peptides, each 20 aminoacids in length. Since T cell sites are muchshorter than this (10-12 amino acids), small T cell sites may be located in the general polymorphicregions without necessarily containing polymorphic residues. Wethus attempted to define precisely the amino and carboxyl terminal boundaries of the T cell site(s) within Th2R(73), the second most immunodominant human T cell domain and one of the regions shown to be polymorphic (28). Initially, three overlapping 12-mer peptides from within Th2Rwere synthesized: 326-337; 330-341; 332-343. Both proliferating and helper T cells from B10.BRand B10.A(4R)mice recognized peptide 326-337, but none of the other peptides. The T cell epitope was further defined by synthesizing amino terminal and carboxyl terminal nested peptides from within peptide 326-337. The amino terminal series revealed that 328-337 had full activity but that peptide 329-337 had only minimal activity; the carboxyl terminal series revealed that peptide 328-334had full activity but that peptide 328-333 had no activity. Thus, the I-Ak-restricted minimal T cell site was328-334,a seven residue T cell site. This is the shortest minimal T cell site, to our knowledge; however, two of the nine known amino acid variations within the CS protein occur within this seven amino acid segment. Wealso located another minimal T cell site within Th2R. Proliferating, but not helper, T cells from B10.D2mice recognized a minimal segment between residues 330 and 343. This minimal T cell site contained three residues knownto be variable. The precise overlap of multiple T sites (Table 3) within a polymorphic segment of the molecule gave even stronger evidence to the contention that variation occurred in response to pressure from immuneT cells. Further confirmation came from the demonstration by de la Cruz et al (74) that T cells primed by one sequence did not recognize variant sequences. Howcould immuneT cells provide pressure for variation? If the final effector mechanismfor sporozoite immunity was mainly antibody, pressure from immuneT cells could not lead to polymorphism.This is because sporozoites bearing new variant T-site sequences wouldbe selected against as strongly as sporozoites bearing nonvariant T-site sequences, by virtue

Annual Reviews MALARIAVACCINE DEVELOPMENT

677

Table3 MinimalT sites within variant region MinimalT Sites

Annu. Rev. Immunol. 1988.6:663-688. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

I-Ak restricted

D K H I E Q Y

I-Ad-restricted

Th2R (7G8)

H I E Q ¥ L K K I K N S I S

H I E Q ¥ L K K I K N S I S

Variant Th2R Variant Th2R (LE5) (Wellcome)

---K ----

----__ ----Q --L --

of their invariant B cell epitope, (NANP)n.In contrast, if the final effector mechanism for sporozoite immunity was mainly cellular, sporozoites bearing new variant T-site sequences would be positively selected. If CD8 (cytotoxic) T cells (CTL) are responsible for sporozoite immunity humans, as they are in mice (49, 50), hepatocytes bearing new variant CTL epitopes would clearly escape destruction and parasite development would continue unabated. As yet, CTL epitopes have not been defined for the CS molecule, but it is likely that such epitopes will share physicochemical properties with helper T-cell epitopes--for example, helical amphipathicity (52)--and thus they may overlap the already defined human helper cell epitopes. Although very few CTLepitopes have been described (53, 54), all are known to conform with the helical amphipathic model. Thus, we believe that selection for polymorphism has probably occurred through immune pressure from cytotoxic T cells (CTL) and, although CTLepitopes have not yet been defined for the CS protein, it is likely that they overlap the already defined major CD4 epitopes which are variant. Variation could thus have a dual effect on parasite survival. First, variant parasites within hepatocytes would escape destruction by CTL and, second, variation within helper T cell sites would result in a lower titer of antisporozoite blocking antibody for any given sporozoite load in the

Annual Reviews 678

GOOD, BERZOFSKY& MILLER

population. This probably explains the low anti-(NANP)nserumtiters observedfor individuals from The Gambia(72).

Annu. Rev. Immunol. 1988.6:663-688. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

Design of the Optimal Antisporozoite

Vaccine

INDUCTION OFBLOCKING ANTIBODY High titer antibody to the repeating epitope in the central third of the CSprotein can prevent sporozoiteinduced infection to a variable degree. In one murine study using P. berghei, already referred to (32), peptide vaccinationled to protection 30%-40% of mice from challenge with 500 sporozoites, but not 1000 sporozoites, while sporozoite immunizationresulted in completeprotection. In anotherstudy with P. berghei, up to 805’oprotection wasachieved using a peptide vaccine(75). In a separate study, Lal et al (76) studied P. yoelii synthetic vaccine. Theyfoundno protection against a challenge from500 sporozoites, eventhoughhigh antibodytiters to sporozoiteswere achieved. To test the hypothesis that lack of immunityindicated escape by parasites bearing variant repeat-epitopes, they checkedsporozoites derived from parasites that appeared in vaccinated mice and found the sameserological epitopes on the sporozoite surface as on the sporozoites used to challenge

the mice. DNAmelting studies

further

indicated

that

parasites whichescapedthe antibody surveillance were not variant with respect to the immunodominant B cell epitope of the CS protein. Other animalvaccine studies, including someusing recombinantvaccinia viruses, are currentlyin progress. Twoindependenthumanvaccine trials haveso far taken place. In both, the first goal of vaccination (Phase 1) was to producehigh titer anti(NANP)n antibody responsesin all the vaccinated individuals. To achieve this goal, the NANP epitope wascovalently fused to a "carrier" epitope, the function of whichwasto stimulate helper T cells to help an anti-NANP B cell response.Onevaccine, already referred to, wasproducedas a fusion protein between NANP-repeatsand part of a sequence encoded by a tetracyclineresistancegeneread out of frame;it wasreferred to as R32tet3~. Fifteen individuals were inoculated repetitively with varying doses of R32tet32(10/~gto 800/~g);alumwasused as an adjuvant(5). Oneindividual whoreceived the highest dose and had the highest antibody response did not develop malaria following sporozoite challenge. The others whohad lower antibody titers did develop malaria. Althoughthe results were encouraging,the first goal--to achieve an antibody response in all the vaccinees--wasnot achieved. As outlined above, both murineand human studies suggest that the poor immunoresponsiveness was the result of immuneresponse (Ir) gene control. Certainly, the (NANP)n epitope was not an immunodominantT cell site in humansfrom The Gambia; it induced a response in only 1 of 35 individuals (72). It would not

Annual Reviews

Annu. Rev. Immunol. 1988.6:663-688. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

MALARIA VACCINE DEVELOPMENT

679

surprising if the "tet32" portion was also not an immunodominant human T cell site. The other humanvaccine study involved covalently attaching (NANP)3 to tetanus toxoid and using alum as an adjuvant (7). Most individuals did not develop a high antibody response, but three individuals who did respond were challenged by the bite of infected mosquitoes. One person did not develop malaria, and the other two whodid had slightly prolonged prepatent periods, indicating that many, but not all, sporozoites were inactivated. Whyso few individuals responded to tetanus toxoid-(NANP)3 is not clear, but prior exposure to tetanus toxoid mayhave limited the response to the conjugated vaccine. One well-described phenomenonin laboratory animals is epitopic suppression (77-80). Prior exposure to carrier protein results in suppression of the antibody response to a novel B cell epitope (which maybe peptide) when the animal is challenged with the carrier protein conjugated to that B cell epitope. If this is the explanation for the poor antibody response in the humantrial, a different carrier may overcome the problem. Of course, if epitopic suppression is responsible for the low response, administering tetanus toxoid-(NANP)3--tochildren in place of tetanus toxoid vaccination, may induce both high titer anti-NANPand anti-tetanus toxoid antibodies. A morecrucial issue is howeffective this antibody will be, once induced. The answer is in part empirical, in that it is not knownwhat percentage of people with high titer antibody will be protected; but the immunobiology of the infection must also be recognized. Sporozoites inoculated by the mosquito are rapidly cleared from the bloodstream, invading hepatic parenchymalcells where they undergo their next proliferative cycle. Once within hepatic cells, antibody to the CS protein no longer affects parasite development. Furthermore, one P. falciparum parasite that escapes antisporozoite immunitycan lead to a fulminant infection as a proliferating asexual erythrocytic parasite. Since immunityinduced against sporozoite antigens is specific to this stage in the life cycle, the asexual infection is unaffected by the vaccine. Thus, boosting the antibody response during infection, if it were to occur, wouldonly affect subsequentinfections. From the studies in rodent models, high titer antibody did not produce protection in all the animals. The protection in mice immunizedwith P. berghei was between 30%and 80%. If, however, a larger inoculum of sporozoites were used in the challenge, then all animals were infected in one of the studies. Furthermore, high titer antibody to the repeating epitopes of P. yoelii did not lead to a significant level of protection. Thus, it appears from the rodent studies, that a vaccine based solely on antibodies to the repeating epitope may not be adequate. It is not known, however, to what extent the fine specificity of the antibody responsewill be critical.

Annual Reviews 680

GOOD, BERZOFSKY & MILLER

Annu. Rev. Immunol. 1988.6:663-688. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

NATURALBOOSTING Another

issue in antibody-based vaccines is whether boosting during infection is a desirable and attainable goal. Whilea vaccine that could not be boosted by natural infection maybe useful for tourists, military personnel, or to control epidemics, it may be impractical for general use in tropical humoral countries where a vaccine is most needed. The only practical humoral vaccine for use in these countries may be one that allows natural boosting by sporozoites themselves. Thus, when an immuneindividual receives sporozoite inoculation, the sporozoites are destroyed, but at the same time they boost the immuneresponse in readiness for the next inoculation. Even then, a vaccinated individual living in an area of seasonal transmission may have low levels of antibody at the beginning of the wet season (when malarial transmission occurs) and may be susceptible to sporozoites before boosting occurs. For natural boosting to occur, the immunogenshould contain a T cell epitope covalently linked to a B cell epitope (81, 82); the two epitopes usually derive from the same molecule in the parasite. This is because the B cell, following surface Ig recognition of the B cell epitope, endocytoses and processes the entire molecule (83). Only if this molecule contains a cell epitope can the B cell present a T cell epitope and attract T cell help. If there is not a population of primedT cells available, T cell help will be limiting. Thus, the vaccine must contain parasite-derived T cell epitopes from the same molecule as the B cell epitope. This approach was followed in constructing a synthetic peptide immunogencontaining a B and T cell epitope from the CS protein, as noted above (64). This dogma, developed from the study of model systems, was recently challenged by investigators studying the immuneresponse to influenza (84). They noted that T cells, primed by the influenza Mprotein, could provide help to B cells that recognized the surface HAprotein. A possible explanation was that B cells recognized a HAepitope on the viral surface, endocytosed and processed the entire virus including the Mprotein, and then attracted T cell help. Perhaps a similar event could occur in the immuneresponse to sporozoites if sporozoites or fragments of sporozoites are endocytosed by B cells. In any case, for natural boosting the T epitope should derive from the sporozoite, if not the CSprotein itself. Becauseof the CS-proteinvariation within majorT sites, significant natural boosting of the antibody response to a vaccine may be unattainable. Because only four CS proteins have been sequenced, it is not knownhow extensive the variation will be, nor do we knowwhether it will be possible to create vaccines containing multiple variant T sites representative of manyvariants within the population of paratites; which would, thus, prime for natural boosting following sporozoite inoculation. If the variation is extensive and natural boosting is not an attainable goal, an alternative is

Annual Reviews

MALARIA VACCINEDEVELOPMENT 681

Annu. Rev. Immunol. 1988.6:663-688. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

to use a repository intermittent release antigen and a conventional"carrier" for Tcell help. CELLULAR IMMUNITY For a sporozoite vaccine reliant on cellular immunity, the mainproblemis to define the correct immunogen(s). This maynot be the CSprotein; however,the observedvariation within T cell domains strongly suggests that the CSprotein is a critical antigen for cellular immunity.Unfortunately,the variation maynullify its use as an effective immunogen. Vaccinationstudies using the entire CSprotein in rodents or monkeys will define whetherit does stimulate protective cellular immunity, providingcloned parasite lines of knownCSprotein sequenceare used for the challenge. If the CSprotein, which is polymorphic, proves to be the correct immunogen for sporozoite immunity, howcan sporozoites successfully immunizeanimals for challenge with the samepopulation of sporozoites?Sporozoitesused for laboratory experimentsare derived from asexual lines that are continuously passaged in non-immuneanimals. Consequently, no mutation pressure is applied to the CS gene and the parasite with the fastest asexual growthrate will dominate.Thus,the CS genemaynot be polymorphic in such artificial situations. This wasindeed found to be the case whende la Cruz et al (22) sequenceda second genederivedfroma P. yoeliiline whoseCSgenehad beenpreviouslycloned (21). In contrast to laboratory experimentswheresporozoite immunity relatively easy to induce, 60 of 83 adult Kenyansfrom a region holoendemicfor P. falciparumdevelopedP. falciparuminfections within 100 days following malaria chemotherapy(37). It is unknown to what degree asexual stage immunityprotected the 23 individuals whodid not develop parasitemia, but clearly, lifelong exposureto sporozoites fromthe field does not render immunityto the local population of parasites. While part of the explanation for lack of immunitywill be due to low Ir gene immunoresponsiveness to given T epitopes, part of the explanationwill be due to parasite polymorphism. The extent of this polymorphism will determinethe feasibility of a sporozoite vaccine based on cellular immunity. Todeterminewhetherthere are other sporozoiteproteins or liver stage antigens that should be lookedfor, an approachis to tolerize miceneonatally to a murine malaria CS protein and then attempt to immunize themwith irradiated sporozoites or non-irradiated sporozoites. If such animalscould be protectively immunized,that wouldbe goodevidencefor the existence of other protective antigens. If the animals could not be immunized,this wouldsuggestthat the CSprotein wasthe critical antigen or a necessary antigen. If neonatally CS-protein tolerized mice could be immunizedwith non-irradiated sporozoites, but not with irradiated

Annual Reviews

Annu. Rev. Immunol. 1988.6:663-688. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

682

GOOD, BERZOFSKY & MILLER

sporozoites, this result wouldsuggest that a developmentalliver stage antigen could confer protection. Havingdefined the correct immunogen to stimulate cellular immunity, it will probablybe necessaryto stimulateCD8cells for protection (49, 50). Generally, a live immunogen,e.g. a recombinantvirus, is required to stimulate CD8cells (CTL).Recently, however,an NS-I fusion protein has been reported capable of stimulating CTL(85), and so have liposomes (86). Suchnonviral vaccines maybe safer but moreexpensivethan viral vectors(87). Apotential difficulty in using a live vector is the risk of serious side effects if administeredto immunosuppressed patients. Theconcurrenceof AIDSand malaria in Africa makesthis a serious problem.An alternative maybe somesynthetic peptide construct containing several CD4and CD8 T cell epitopes and a segmentof (NANP)n repeats. Thechoice of adjuvant maybe critical to achieve a high antibody response and to induce CTL. Alummaynot be suitable, and moreexperimental adjuvants, e.g. liposomes (86), ISCOMS (immunestimulatory complexes) (88), or muramyl dipeptide derivatives (89), maybe necessary. Amajor problemto overcome will be, of course, the paucity of human T cell epitopes on tl~e CSmolecule (14 individuals from 35 tested in The Gambiadid not respond to any of the synthetic peptides that spannedthe entire CSmolecule)(72); another problemis parasite variation within the immunodominant T cell epitopes. In mice, low responsivenessto T cell epitopes can be overcomeby administering interleukin-2 (IL-2) with the antigen (90). Wehaverecently firmedthis finding using CSprotein-derivedT cell epitopes (91). Such approach maybe useful in humans. To overcomeproblems of parasite variation, a vaccine will need to contain as manyvariant sequencesas possible. Eventhen, a numberof individuals will not respondto any of the variant sequences,unless special adjuvants,for exampleIL-2, are used. Someof these problems are not unique to malaria sporozoite vaccine developmentand are being addressed in other vaccine programs(reviewed in 92). Conclusions Thedesign of a subunit peptide vaccine requires knowledge of the effector mechanismsand the immunogens for induction of these effector mechanisms. For antibody-mediatedmechanisms,only the B epitope need come from the pathogen.It is usually assumedthat the T epitopes for help in antibody productionshould also be derived fromthe pathogen,since this allowsfor boostingduringinfection. Malariaparasites, however,are only affected by antisporozoite antibodies during the first few minutesafter the mosquitoinoculates the malarial parasites into humans.Boostingof

Annual Reviews

Annu. Rev. Immunol. 1988.6:663-688. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

MALARIA VACCINE DEVELOPMENT

683

antibody wouldbe protective only against a subsequentinfection. Furthermore,if the T epitopes are variant, as is the case with the CSprotein, then the chance of boosting from any particular infection is reduced. Perhapssporozoite proteins other than the CS protein can supply helper T cell epitopes for antibodyto the CSprotein. Thequestion still remains, howcan wedesign a vaccine that maintainsa high level of antisporozoite antibody, especially in a populationin the developingworldwhereit is difficult andexpensiveto give repeatedimmunizations? A repository antigenthat is releasedat intervals is onesolution. A sporozoite vaccine based on antibody alone maynot be the most effective. In rodent malaria, the highest level of immunityis inducedby immunization with irradiated sporozoites. T cells, not antibody, transfer immunityfrom these immunizedanimals to naive mice. Furthermore, ablation of CD8cells converts themfrom protected to fully susceptible mice. This suggests that cytotoxic cells mediateparasite killing, perhaps throughan effect on infected liver cells. What,then, is the target antigen of this immunity?The regions of the CS protein most immunogenicfor CD4cells are variant. Since selection of variation in the CSprotein is likely to be againstan effector epitope,it is possiblethat these sameregions contain epitopes for CD8,cytotoxic T cells. Thechallengeis to identify invariant parasite epitopeson infected liver cells that can be recognizedby T cells. This mayrequire fresh approachesto the study of immunityto the sporozoiteand liver stages. Despite the remainingobstacles, the progress has beenrapid since the discoveryof the CSprotein by the Nussenzweigs and their colleaguesin 1980(14). Theseriousnessof malariain the developingworld demands that wecontinueuntil an effective vaccine is found. ACKNOWLEDGMENTS

Dr. F. A. Nevais thanked for continued support. Wethank W.Weiss, V. F. de la Cruz, D. Pombo,A. Lal, T. F. McCutchan, K. Cease, H. Margalit, and J. Cornette for advice and suggestions. M. F. Goodreceived partial support from a Neil HamiltonFairley Fellowship from National Health and MedicalResearchCouncil (Australia) and from a Fulbright Award. Literature Cited 1. Miller, L. H., Howard,R. J., Carter, R., Good, M. F., Nussenzweig, V., Nussenzweig, R. S. 1986. Research toward malaria vaccines. Science 234:1349-56 2. Desowitz, R. S., Miller, L. H. 1980. A perspective on malaria vaccines. Bull. WHO6:897-908 3. Trager, W., Jensen, J. B. 1976. Human

malaria parasites in continuous culture. Science 193:673-75 4. Collins, W. E., Anders, R. F., Pappaioanou, M., Campbell, G. H., Brown, G. V., Kemp,D. J., Coppel, R. L., Skinner, J. C., Andrysiak, P. M., Favaloro, J. M., Corcoran, L. M., Broderson, J. R., Mitchell, G. F., Campbell, C. C.

Annual Reviews

Annu. Rev. Immunol. 1988.6:663-688. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

684

GOOD, BERZOFSKY& MILLER 1986. Immunizationof Aotus monkeys 1985. Circumsporozoite proteins of with recombinantproteins of an erythmalariaparasites. Cell 42:401-3 rocyte surface antigen of Plasmodium 14. Yoshida, N., Nussenzweig,R. S., Potfalciparum. Nature 323:259q52 ocnjak, P., Nussenzweig,V., Aikawa, 5. Ballou, W.R., Hoffman,S. L., SherM. 1980. Hybridomaproduces protecwood,J. A., Hollingdale, M. R., Neva, tive antibodiesdirected against the spoF. A., Hockmeyer,W. T., Gordon, D. rozoite stage of malariaparasites. SciM., Schneider,I., Wirtz, R. A., Young, ence 207:71-73 J. F., Wasserman,G. F., Reeve, P., 15. Potocnjak, P., Yoshida, N., NussenDiggs,C. L., Chulay,J. D. 1987.Safezweig, R. S., Nussenzweig,V. 1980. ty and efficacy of a recombinant Monovalentfragments (Fab) of monoDNAPlasmodium falciparum sporoclonal antibodiesto a sporozoitesurface zoite vaccine.LancetI: 1277-81 antigen (Pb44) protect mice against 6. Patarroyo, M. E., Romero,P., Torres, malarial infection. J. Exp. Med. 151: M. L., Clavijo, P., Moreno, A., 1504-13 Martinez, A., Rodriguez, R., Guzman, 16. Ellis, J,, Ozaki, L. S., Gwadz,R. W., F., Cabezas,E. 1987. Inductionof proCochrane,A. H., Nussenzweig,V., Nustective immunityagainst experimental senzweig, R. S., Godson,G. N. 1983. infection with malaria using synthetic Cloning and expression of the Plaspeptides. Nature328:629-31 modiumknowlesi sporozoite surface 7. Herrington,D. A., Clyde,D. F., Losonantigen in E. coli. Nature302:536-38 sky, G., Cortesia, M., Murphy,F. R., 17. Sharma,S., Svec, P., Mitchell, G. H., Davis, J., Baqar, A., Felix, A. M., Godson, G. N. 1985. Diversity of Heimer,E. P., Gillessen, D., Nardin,E., circumsporozoite antigen genes from Nussenzweig,R. S., Nusssenzweig,V., twostrains of the malarialparasite PlasHollingdale,M.R., Levine, M.M. 1987. modiumknowlesi. Science 229:779-82 Safety and immunogenicityin manof 18. Galinski, M. R., Arnot, D. E., Coa synthetic peptide malaria vaccine chrane, A. H., Barnwell,J. W., Nussenagainst Plasmodiumfalciparum sporozweig,R. S., Enea, V, 1987.Thecircumzoites. Nature328:257-59 sporozoite gene of the Plasmodium 8. Dame,J. B., Williams, J. L., McCutcynomolgicomplex.Cell 48:311-19 chan, T. F., Weber,J. L., Wirtz, R. A., 19. Eichinger, D. J., Arnot, D. E., Tam,J. Hockmeyer, W. T., Maloy, W. L., P., Nussenzweig,V., Enea, V. 1986. Haynes,J. D., Schneider, I., Roberts, Circumsporozoite protein of PlasmoD., Sanders,G. S., Reddy,E. P., Diggs, diumberghei: genecloning and identiC. L., Miller, L. H. 1984. Structure of fication of the immunodominant epithe gene encoding the immunodominant topes. Mol.Cell. Biol. 6:3965-72 surface antigen on the sporozoiteof the 20. Weber,J. L., Egan,J. E., Lyon,J. A., humanmalaria parasite Plasmodium Wirtz, R. A., Charoenvit,Y., Maloy,W. falciparum. Science 225:593-99 L., Hockmeyer,W.T. 1987. Plasmodium 9. Aley, S. B., Bates, M.D., Tam,J. P., berghei: cloningof the circumsporozoite Hollingdale,M.R. 1986.Synthetic pepprotein gene. Exp. Parasitol. 63: 295tides fromthe circumsporozoiteproteins 300 of Plasmodiumfalciparum and Plas- 21. Lal, A. A., de la Cruz, V. F., Welsh, modiumknowlesi recognize the human J. A., Charoenvit, Y., Maloy,W. L., hepatomacell line HepG2-A16in vitro. McCutchan, T. F. 1987. Structure of the J. Exp. Med. 164:1915-22 gene encoding the circumsporozoite 10. Meis, J. F. G. M., Verhave,J. P., Jap, protein of Plasmodium yoelii. A rodent P. H. K., Meuwissen, J. H. E. Th. 1983. modelfor examiningantimalarial sporoAnultrastructural study on the role of zoite vaccines.J. Biol. Chem.262: 2937Kupffercells in the processof infection 40 by Plasmodiumberghei sporozoites in 22. de la Cruz, V. F., Lal, A. A., McCutrats. Parasitology86:231-42 chart, T. F. 1987. Variation among 11. Mulligan,H. W., Russell, P. F., Mohan, circumsporozoiteprotein genes from roB. N. 1941. Active immunization of dent malarias. Mol. Biochem.Parasitol. fowls against Plasmodiumgallinaceum In press by injections of killed homologous 23. Enea,V., Ellis, J., Zavala,F., Arnot,D. sporozoites. J. Mal.Inst. India 4:25-34 E., Asavanich,A., Masuda,A., Quakyi, 12. Nussenzweig, R. S., Nussenzweig,V. I., Nussenzweig,R. S. 1984. DNA clon1984. Developmentof sporozoite vacing of Plasmodiumfalciparum circumcines. Philos. Trans.R. Soc, London Ser. sporozoite gene: aminoacid sequenceof B 307:117-28 repetitive epitope. Science225:628-30 13. Nussenzweig, V., Nussenzweig,R. S. 24. Arnot, D. E., Barawe11,J. W., Tam,J.

Annual Reviews

Annu. Rev. Immunol. 1988.6:663-688. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

MALARIAVACCINE DEVELOPMENT

685

P., Nussenzweig, V., Nussenzweig, R. S., 33. Weber,J. L., Hockmeyer,W.T. 1985. Structure of the circumsporozoiteproEnea, V. 1985. Circumsporozoiteprotein gene in I8 strains of Plasmodium tein of Plasmodium vivax: gene cloning falciparum.Mol.Biochem.Parasitol. 15: and characterization of the immunodominantepitope. Science 230:815-18 305-16 34. Zavala, F., Masuda,A., Graves, P. M., 25. McCutchan,T. F., Lal, A. A., de la Nussenzweig,V., Nussenzweig,R. S. Cruz,V. F., Miller, L. H., Maloy,W.L., 1985.Ubiquityof the repetitive epitope Charoenvit, Y., Beaudoin, R. L., of the CSprotein in different isolates of Guerry,P., Wistar, R. Jr., Hoffman,S. L., Hockmeyer,W.T., Collins, W.E., humanmalaria parasites. J. Immunol. Wirth, D. 1985. Sequenceof the immu135:2790-93 35. Lawler,J., Hynes,R. O. 1986.Thestrucnodominant epitope for the surface protein on sporozoites of Plasmodium ture of human thrombospondin, an vivax. Science 230:1381-83 adhesiveglycoproteinwith multiplecalcium-bindingsites and homologieswith 26. Lockyer, M. J., Schwarz, R. T. 1987. Strain variation in the circumsporozoite several different proteins. J. Cell Biol. protein geneof Plasmodium falciparum, 103:1635.48 MoLBiochem.Parasitol. 22:101-8 36. Spitalny, G. L., Rivera-Oritz, C.-I., Nussenzweig,R. S. 1976. Plasmodium 27. de la Cruz, V. F., Lal, A. A., Welsh,J, ber#hei:the spleenin sp0rozoite-induced A., McCutchan,T. F. 1987. Evolution of the immunodominant domainof the immunityto mousemalaria. Exp. Paracircumsporozoite protein gene from sitoL 40:179-88 Plasmodium vivax: implications for vac- 37. Hoffman,S. L., Oster, C. N., Plowe,C. V., Woollett,G.R., Beier, J. C., Chulay, cines. J. Biol. Chem.262:6464~67 J. D., Wirtz,R. A., Hollingdale,M.R., 28. de la Cruz, V. F., Lal, A. A., McCutchan, T. F. 1987. Sequencevariation in Mugambi,M. 1987. Naturally acquired putative functional domains of the antibodiesto sporozoitesdo not prevent circumsporozoiteprotein of Plasmodium malaria: vaccine development implifalciparum: implications for vaccine cations. Science 237:639-42 development.J. Biol. Chem.262:11935- 38. Chen, D. H., Tigelaar, R. E., Wein39 baum, F. I. 1977. Immunityto sporo29. Young,J. F., Hockmeyer, W.T., Gross, zoite-inducedmalariainfection in mice. M.Ballou, W.R., Wirtz,R. A., Trosper, I. Theeffect of immunizationof T and J. H., Beaudoin,R. L., Hollingdale,M. B cell-deficient mice. J. Immunol.118: R., Miller, L. H., Diggs,C. L., Rosen1322-27 berg, M. 1985. Expression of Plas- 39. Spitalny, G. L., Verhave, J. P., modiumfalciparum circumsporozoite Meuwissen,J. H. E. Th., Nussenzweig, proteinsin Escherichia coil for potential R. S. 1977. Plasmodium berghei: T cell use in a humanmalaria vaccine. Science dependence of sporozoite-induced 228:958.62 immunityin rodents. Exp. Parasitol. 42: 30. Ballou, W.R., Rothbard,J., Wirtz, R. 73-81 A., Gordon,D. M., Williams, J. S., 40. Verhave,J. P., Strickland,G. T., Jaffe, Gore,R. W., Schneider,I., Hollingdale, H. A., Ahmed,A. 1978. Studies on the M. R., Beaudoin,R. L., Maloy,W.L., transfer of protective immunitywith Miller, L. H., Hockmeyer,W.T. 1985. lymphoid cells from mice immuneto Immunogenicityof synthetic peptides malaria sporozoites. J. lmmunoL121: from circumsporozoiteprotein of Plas1031-33 modiumf alciparum.Science 228:996-99 41. Spitalny, G. L., Nussenzweig,R. S. 31. Zavala, F., Tam,J. P., Hollingdale,M. 1973. Plasmodium berghei: relationship R., Cochrane,A. H., Quakyi,I., Nusbetweenprotective immunityand antisenzweig,R. S., Nussenzweig, V. 1985. sporozoite (CSP)antibodyin mice. Exp. Rati.onalefor development of a synthetic Parasitol. 33:168-78 vaccine against Plasmodium falciparum 42. Clyde, D. F., McCarthy,V. C., Miller, malaria. Science 228:1436-40 R. M., Hornick,R. B. 1973.Specificity 32. Egan,J. E., Weber,J. L., Ballou,W.R., of protection of manimmunizedagainst Hollingdale, M. R., Majarian, W.R., sporozoite-inducedfalciparummalaria. Gordon, D. M., Maloy, W.L., HoffAm. J. Med.Sci. 266:398-403 man,S. L., Wirtz,R. A., Schneider,I., 43. Cochrane,A. H., Aikawa,M., Jeng, M., Woollett, G. R., Young,J. F., HockNussenzweig, R. S. 1976. Antibodymeyer, W.T. 1987. Efficacy of murine induced ultrastructural changes of malaria sporozoite vaccines: implimalarial sporozoites. J. Immunol.116: cations for humanvaccine development. 859-67 Science 236:453-56 44. Zavala, F., Cochrane,A. H., Nardin,E.

Annu. Rev. Immunol. 1988.6:663-688. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

Annual Reviews 686 GOOD, BERZOFSKY& MILLER H., Nt~ssenzweig,R. S., Nussenzweig, Jordan,B. R., Cerottini, J.-C. 1986.H-2V. 1983. Circumsporozoiteproteins of restricted cytolyticcells specificfor HLA malaria parasites contain a single can recognize a synthetic HLApeptide. immunodominantregion with two or Nature 324:578-79 moreidentical epitopes. J. Exp. Med. 55. Tite, J. P., Janeway,C. A. Jr. 1984. 157:1947-57 Clonedhelper T cells can kill B lym45, Ferreira, A., Schofield, L., Enea, V., phomacells in the presenceof specific Schellekens, H., Van Der Meide, P., antigen: Ia restriction and cognatevs. Collins, W.E., Nussenzweig,R. S., noncognateinteractions in cytolysis. Nussenzweig,V. 1986. Inhibition of Eur. J. Immunol.14:878-86 developmentof exoerythrocytic forms 56. Ozaki, S., York-Jolley, J., Kawamura, H., Berzofsky,J. A. 1987. Clonedproof malaria parasites by ~,-interferon. tein antigen-specific,Ia-restrictedT cells Science 232:881-84 46. Maheshwari,R. K., Czarniecki, C. W., withbothhelperandcytolytic activities: mechanismof activation and killing. Dutta, G. P., Puri, S. K., Dhawan,B. Cell. lmmunol.105:301-16 N., Friedman, R. M. 1986. Recombinant human gammainterferon in57. Reinherz, E. L., Morimoto,C., Fitzgerald, K. A., Hussey,R. E., Daley,J. hibits simianmalaria,lnfect, lrnmun.53: F., Schlossman, S. F. 1982. Hetero628-29 geneity of humanT4+ inducer T cells 47. Aikawa,M., Yoshida, N., Nussenzweig, defined by a monoclonal,tntibody that R. S., Nussenzweig,V. 1981. The prodelineates two functional subpopulatective antigen of malarial sporozoites tions, J. Immunol.128:463-68 (Plasmodium beryhei)is a differentiation antigen. J. Immunol.126:2494-95 58. Arthur, R. P., Mason,D. 1986. T cells that helpB cell responsesto solubleanti48. Morrison, L. A., Lukacher, A. E., gen are distinguishable from those proBraciale,V.L., Fan,D.P., Braciale,T. J. ducing interleukin 2 on mitogenic or 1986.Differencesin antigenpresentation allogeneicstimulation.J. Exp.Med.163: to MHC class I- and class II-restrieted 774-86 influenza virus-specific cytolytic T lymphocyteclones. J. Exp. Med.163: 59. Mosmann, T. R,, Cherwinksi,H., Bond, M. W., Giedlin, M.A., Coffman,R. L. 903-21 1986. Twotypes of murinehelper T cell 49. Weiss, W.R., Sedegah, M., Beaudoin, clone I. Definitionaccordingto profiles R. L., Miller, L. H., Good,M. F. 1988. ÷ of lymphokineactivities and secreted CD8 T cells (cytotoxic/suppressors) are proteins. J. Immunol.136:2348-57 required for protection in mice immunized with malaria sporozoites. Proc. 60. Good,M. F., Berzofsky, J. A., Maloy, W.L., Hayashi, Y., Fujii, N., HockNatl. Acad. Sci. USA85:573-76 meyer,W.T., Miller, L. H. 1986.Genetic 50. Schofield,L., Villaquiran,J., Ferreira, A., Schellekens,H., Nussenzweig, R. S., control of the immune response in mice to a Plasmodium falciparum sporozoite Nussenzweig, V. 1988. Gammaintervaccine. Widespreadnonresponsiveness feron, CD8T cells and antibodies required for immunityto malariasporoto single malaria T epitope in highly repetitive vaccine. J. Exp. Med.164: zoites. Nature. 655-60 51. Buller, R. M.L., Holmes,K. L., Hiigin, A., Frederickson, T. N., Morse,H. C. 61. Del Giudice,G., Cooper,J. A., Merino, J., Verdini, A. S., Pessi, A., Togna,A. Ill 1987. Induction of cytotoxic T-cell R., Engers, H. D., Corradin, G., responsesin vivo in the absenceof CD4 Lambert, P.-H. 1986. The antibody helper ceils. Nature328:77-79 responsein miceto carrier-free synthetic 52. Berzofsky,J. A., Cease,K. B., Cornette, polymers of Plasmodiurn falciparum J. L., Sponge,J. L., Margalit, H., Bercircumsporozoite repetitive epitopeis Ikower,I. J., Good,M. F., Miller, L. A~-restricted: possible implicationsfor H., DeLisi, C. 1987, Protein antigenic structures recognizedby T cells: potenmalariavaccines, d. lmmunol.137: 295255 tial applications to vaccine design. 62. Togna,A. R., Del Giudice,G., Verdini, lmrnunol. Rev. 98:9-52 A.S., Bonelli,F., Pessi, A., Engers,H. 53. Townsend, A. R. M., Rothbard, J., D., Corradin,G. 1986. Synthetic P.falGotch, F. M., Bahadur,G., Wraith, D., ciparumcircumsporozoitepeptideselicit McMichael,A. J. 1986. Theepitopes of ÷ T cell proliferative heterogenousL3T4 influenza nucleoprotein recognized by ~ mice.J. Immunol.137: responses in H-2 cytotoxic T lymphocytescan be defined 2956-60 with short synthetic peptides. Cell 44: 63. Good,M. F., Berzofsky, J. A., Maloy, 959-68 W.L., Lunde,M. N., Moss,B., Miller, 54. Maryanski, J. L., Pala, P., Corradin,G.,

Annual Reviews

Annu. Rev. Immunol. 1988.6:663-688. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

MALARIAVACCINE DEVELOPMENT 687 L. H., Hayashi, Y., Fujii, N., Hock- 75. Zavala, F., Tam,J. P., Barr, P. J., meyer,W.T. 1987. Genetic control of Romero,P. J., Ley, V., Nussenzweig, the immuneresponse to a Plasmodium R. S., Nussenzweig,V. 1987. Synthetic falciparumsporozoitevaccine andto the peptide vaccine confers protection circumsporozoiteprotein. In Vaccines against murine malaria. J. Exp. Med. 87, ed. R. M. Chanock,R. A., Lerner, 166:1591-96 F. Brown,H. Ginsberg, pp. 81-85. New 76. Lal, A. A., de la Cruz, V. F., Good,M. York: Cold Spring Harbor Lab. F., Weiss, W.J., Lunde,M., Maloy,W. 64. Good,M. F., Maloy,W.L., Lunde, M. L., Welsh,J. A., McCutchan, T. F. 1987. Testingsporozoite-derivedmalarial vacN., Margalit,H., Cornette,J. L., Smith, G. L., Moss,B., Miller,L. H., Berzofsky, cines in vivo using a rodent malaria J. A. 1987. Construction of synthetic system. Proc. Natl. Acad.Sci. USA84: immunogen: use of newT helper epitope 8647-51 on malaria circumsporozoite protein. 77. Herzenberg,L. A., Tokuhisa,T. 1982. Science 235:1059-62 Epitope-specificregulation. I. Carrier65. DeLisi,C., Berzofsky,J. A.1985.T cell specific inductionof suppressionfor IgG antigenic sites tend to be amphipathic anti-hapten antibodyresponses. J. Exp. structures. Proc. Natl. Acad.Sci. USA Med. 155:1730-40 82:7048-52 78. Herzenberg,L. A., Tokuhisa,T., Parks, 66. Spouge,J. L., Guy,H. R., Cornette, J. D. R., Herzenberg,L. A. 1982. EpitopeL., Margalit, H., Cease,K., Berzofsky, specific regulation. II. Abistable, IghJ. A., DeLisi, C. 1987. Strong conrestricted regulatory mechanism central formationalpropensities enhanceT cell to immunologicmemory.J. Exp. Med. antigenicity. J. lmmunol.138:204-12 155:1741-53 67. Margalit,H., Spouge,J. L., Cornette,J. 79. Jacob, C. O., Arnon,R., Sela, M. 1985. L., Cease,K. B., DeLisi,C., Berzofsky, Effect of carrier on the immunogenic J. A. 1987. Prediction of immunocapacity of synthetic cholera vaccine. dominanthelper T cell antigenic sites Mol. Immunol.22:1333-39 from the primarysequence.J. Immunol. 80. Schutze,M.-P.,Leclerc,C., Vogel,F. R., 138:2213-29 Chedid,L. 1987.Epitopicsuppressionin 68. Pombo,D., Maloy,W.L., Berzofsky,J. synthetic vaccinemodels:analysis of the A., Miller, L. H., Good,M. F. 1987. effector mechanisms.Cell. lmmunol. Unpublished 104:79-90 69. Good,M. F., Pombo,D., Houghten,R. 81. Miller,J. F. A.P., Mitchell,G. F. 1969. A., Berzofsky,J. A., Miller, L. H. 1987. Thethymusand the precursors of antiUnpublishedobservations genreactive cells. Nature216:659-63 70. Lamb,J. R., Eckels, D. D., Lake, P., 82. Mitchison,N. A. 1971.Thecarrier effect Woody,J. N., Green, N. 1982. Human in the secondaryresponseto hapten-proT cell clones recognizechemicallysynrein conjugates.II. Cellularcooperation. thesized peptides of influenza hemEur. J. Irnmunol.1:18-27 agglutinin. Nature300:66-69 83. Lanzavecchia,A. 1985. Antigen-specific interaction between T and B cells. 71. Hurwitz,J. L., Heber-Katz,E., Hackett, C. J., Gerhard, W.J. 1984. CharacNature 314:537-39 terization of the murineTHresponseto 84. Scherle, P. A., Gerhard,W.1986. Funcinfluenza virus hemagglutinin:evidence tional analysis of influenza-specific for three majorspecificities. J. lmmunoL helperT cell clonesin vivo. T cells specific 133:3371-77 for internal viral proteins providecog72. Good,M. F., Pombo,D., Quakyi,I. A., nate help for B cell responses to hemRiley, E. M., Houghten,R. A., Menon, agglutinin. J. Exp. Med.164:1114-28 A., Ailing, D. W., Berzofsky, J.A., 85. Yamada,A., Ziese, M. R., Young,J. Miller, L. H. 1988. Human T cell recogF., Yamada,Y. K., Ennis, F. A. 1985. nition of the circumsporozoiteprotein Influenza virus hemagglutinin-specific of Plasmodiumfalciparum. Immunocytotoxic T cell response induced by dominant T cell domains map to the polypeptide produced in Escherichia polymorphicregions of the molecule. coli. J. Exp. Med.162:663-74 Proc. Natl. Acad.Sci. USAIn press 86. Watari,E., Dietzschold,B., Szokan,G., 73. Good,M. F., Pombo,D., Maloy,W.L., Heber-Katz,E. 1987. A synthetic pepde la Cruz,V. F., Miller, L. H., Berzoftide induces long-term protection from sky, J. A. 1987. Parasite polymorphism lethal infectionwithherpessimplexvirus present within minimalT cell ep.itopes 2. J. Exp. Med.165:459-70 of Plasmodium falciparum clrcum87. Cheng,K.-C., Smith, G. L., Moss,B., sporozoiteprotein. J. Immunol.In press Zavala, F., Nussenzweig, R., Nus74. de la Cruz, V. F. 1988. Unpublished senzweig,V. 1986.Expressionof malaria

Annual Reviews

Annu. Rev. Immunol. 1988.6:663-688. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

688

GOOD, BERZOFSKY& MILLER

circumsporozoiteprotein and hepatitis90. Kawamura, H., Rosenberg, S. A., Bvirus surfaceantigenbyinfectiousvacBerzofsky, J. A. 1985. Immunization with antigen and interleukin 2 in vivo cinia virus. In Vaccines86, ed. F. Brown. R. M. Chanock,R. A. Lerner, pp. 165 overcomesIr gene low responsiveness. 68. NewYork: Cold Spring Harbor Lab. J. Exp. Med.162:381-86 88. Morcin,B., Sundquist,B., Hoglund,S., 91. Good,M. F., Pombo,D., Lunde, M. N., Dalsgaard, K., Osterhaus, A. 1984. Maloy, W.L., Halenbeck, R., Koths, Iscom,a novelstructure for antigenpreK., Miller, L. H., Berzofsky,J. A. 1988. sentation of membraneproteins from Recombinanthumaninterleukin-2 (ILenvelopedviruses. Nature308:457-60 2) overcomesgenetic non-responsive89. Stevens, V. C. 1986. Use of synthetic ness to malaria sporozoite peptides. peptides as immunogens for developing Correlation of effect with biological a vaccine against humanchorionic gonactivity of IL-2. Manuscriptsubmitted adotropin. In Synthetic Peptides As 92. Ada, G. L., Jones, P. D. 1987. Vaccines for the future--an update. Immunol. Antigens, ed. R. Porter, J. Whelan,pp. 200-16. Avon,England: Bath Cell Biol. 65:11-24

Annu. Rev. Immunol. 1988.6:663-688. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.

Annu. Rev. Immunol. 1988.6:663-688. Downloaded from arjournals.annualreviews.org by HINARI on 08/29/07. For personal use only.